Compounds and use thereof in methods of treatment

ABSTRACT

Disclosed are compounds of a formula provided herein that show efficacy in the inhibition of SOX18 protein activity, and in particular with respect to the ability of SOX18 to bind DNA and/or particular protein partners. Further, methods of treating angiogenesis- and/or lymphangiogenesis-related diseases, disorders or conditions, such as cancer metastasis and vascular cancers, are provided herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/741,065, filed May 10, 2022, which is a continuation of U.S. patentapplication Ser. No. 16/472,882, filed Jun. 23, 2019, which is a 371national stage application of International Pat. Appl. No.PCT/AU2017/051439, filed Dec. 21, 2017, which claims priority toAustralian Pat. Appl. No. 2016905362, filed Dec. 23, 2016.

REFERENCE TO A SEQUENCE LISTING

This application contains references to amino acid and/or nucleic acidsequences which have been submitted as the sequence listing XML fileentitled “FBR-0006USSeq.IDNos. 1-3”, file size 6.45 KiloBytes (KB),created 6/25/2023, which is hereby incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The invention relates to the field of medical treatment. Moreparticularly, this invention relates to compounds for use in theinhibition of SOX18 transcription factor activity.

BACKGROUND TO THE INVENTION

Any reference to background art herein is not to be construed as anadmission that such art constitutes common general knowledge inAustralia or elsewhere.

Direct modulation of transcription factors (TF) by small moleculesremains a long-standing quest. Early results were limited to nuclearreceptors, which contain a ligand-binding domain targetable by smallmolecules. These findings have been translated into therapeuticapplications in hormone-dependent cancers (Perissi and Rosenfeld, 2005).The current challenge is to reach beyond nuclear receptors to a broaderrange of transcription factors that lack binding pockets for smallmolecular drugs. The task is made difficult due to the lack of definedthree-dimensional structures for many TFs, especially theirprotein-protein binding domains, the difficulty to recombinantly expressTFs, and the lack of assay technology to investigate their mode ofaction (Fontaine et al., 2015). Modulation of TF activity are generallyachieved by changing their gene expression levels or concentration inthe nucleus, or by changing their binding abilities to either DNA orpartner proteins, with the latter the more promising strategy to achieveTF selectivity. A few publications on small molecules disrupting TFrecruitment of partner proteins are a testament to the potential of thisapproach (Miyoshi et al., 2011, Vassilev et al., 2004, Filippakopouloset al., 2010, Vogler et al., 2009, Liu et al., 2014).

Amongst TFs in the human genome, developmental TFs stand out asattractive molecular targets since their expression is oftendysregulated under specific pathological conditions in adult, whilesilenced under physiological conditions (e.g. not required for phenotypemaintenance at adulthood) (Boyadjiev and Jabs, 2000, Darnell, 2002,Lopez-Bigas et al., 2006, Vaquerizas et al., 2009). One class ofdevelopmental factors, the SOX (SRY-related HMG-box) TFs, have recentlyemerged as key regulators of stem cell programming as well as molecularswitches in cancer related conditions (Sarkar and Hochedlinger, 2013,Niwa et al., 2009). Previous attempts at targeting SOX proteins havemainly focused on SOX2 (Narasimhan et al., 2011), a potential oncogenein various cancers (Bass et al., 2009), and SOX18 (Klaus et al., 2016),a key molecular switch for vascular development (Cermenati et al., 2008,Francois et al., 2008, Pennisi et al., 2000). Dawson polyoxometalateshave been shown to inhibit SOX2 DNA-binding, however, only displayed lowselectivity against various TF families, and were never tested in any invitro or in vivo functional assay (Narasimhan et al., 2014, Narasimhanet al., 2011). More recently, SOX DNA decoys have been used as selectiveinhibitors of SOX18 DNA-binding and SOX18-dependent transactivation.While these decoys display great selectivity over non-SOX TF, theycannot diffuse through cell membranes on their own, limiting their scopeof application (Klaus et al., 2016). An unexplored aspect of thepharmacological modulation of SOX TF's is related to how these proteinsrecruit their partners and consequently modulate transcription via arange of protein-protein interactions (PPIs). Arguably, syntheticlibraries do not have the structural diversity required to target PPIs(Hopkins and Groom, 2002, Feher and Schmidt, 2003).

The SOXF group (SOX7, -17 and -18) of transcription factors (TFs) arekey regulators of endothelial cell differentiation during development(Francois et al. 2008, Corada et al. 2013, Hosking et al. 2009, Matsuiet al. 2006, Cermenati et al. 2008, Herpers et al. 2008), and are thuscritical for the formation of vasculature. Mutation or deletion of SoxFgenes compromises arteriovenous specification, blood vascular integrityand lymphangiogenesis, and inhibits tumour growth and metastasis inanimal models of cancer (Duong et al. 2012, Yang et al. 2013, Zhang etal. 2009, Young et al. 2006). More recently, high levels of SOX18 havebeen associated with poor prognosis for cancer in human patients (Eom etal. 2012, Pula et al. 2013, Jethon et al. 2015). Pharmacologicalinhibition of SOX18 protein function therefore presents a potentialavenue for management of the vascular response in cancer as well as apotential therapeutic target in vascular cancers.

Accordingly, there remains a need for compounds that inhibit SOX18protein activity, such as by binding directly thereto, or in proximityto its DNA-binding domain, so as to perturb, for example, SOX18-proteinpartner recruitment and/or SOX18 DNA binding.

SUMMARY OF INVENTION

The present invention is predicated, at least in part, on the findingthat certain compounds of the formula provided herein have efficacy inthe inhibition of SOX18 protein activity, and in particular with respectto the ability of SOX18 to bind DNA and/or particular protein partners.By extension, these compounds are further shown to be effective intreating angiogenesis- and/or lymphangiogenesis-related diseases,disorders or conditions, such as cancer metastasis and vascular cancers.

In a first aspect of the invention is provided a compound of formula(I), or a pharmaceutically acceptable salt, solvate or prodrug thereof:

wherein,

-   -   R₁ is selected from the group consisting of OH and OR₆ wherein        R₆ is C₁-C₄ alkyl;    -   R₂ is selected from the group consisting of H, COORS, and        C(O)NR₈R₉ wherein R₇, R₈ and R₉ are independently selected from        H and C₁-C₄ alkyl;    -   R₃ is L-A wherein L is a linker selected from C₂-C₈ alkyl, C₂-C₈        alkenyl and C₂-C₈ alkoxyalkyl and A is selected from optionally        substituted phenyl and optionally substituted napthyl;    -   R₄ is selected from the group consisting of H, OR₁₀, halo and        C₁-C₄ alkyl wherein R₁₀ is selected from H and C₁-C₄ alkyl; and    -   R₅ is selected from the group consisting of H, OR₁₁, halo and        C₁-C₄ alkyl wherein R₁₁ is selected from H and C₁-C₄ alkyl,    -   wherein, the compound is for use in the inhibition of a SOX18        activity.

In embodiments, R₁ is selected from the group consisting of OH and OMe.

Suitably, R₂ is selected from the group consisting of H, COOH, COOMe and

Preferably, R₂ is selected from COOH and

In embodiments, R₄ is selected from the group consisting of H, OH, OMe,Cl and Me.

Suitably, R₅ is selected from the group consisting of H, OH and OMe.

In certain embodiments, R₄ and R₅ are H.

In embodiments, L is a linker selected from C₂-C₆ alkyl, C₂-C₆ alkenyland C₂-C₆ alkoxyalkyl.

In any of the recited embodiments, R₃ is selected from the groupconsisting of:

wherein, the broken line indicates the attachment from that adjacentatom to the ring of formula I and the structures shown include E/Zisomers thereof.

In one embodiment, the compound of the first aspect is selected from thegroup consisting of:

More preferably, the compound of the first aspect is selected from thegroup consisting of:

Suitably, with respect to the compound of the present aspect, the SOX18activity includes contacting and/or binding to a DNA sequence and/or aprotein. Preferably, the protein is selected from the list consisting ofSOX7, RBPJ, XRCC5, SOX18, ILF3, DDX17 and any combination thereof.

In a second aspect of the invention is provided a pharmaceuticalcomposition comprising a compound of the first aspect, or apharmaceutically acceptable salt, solvate or prodrug thereof, and apharmaceutically acceptable carrier, diluent and/or excipient.

In a third aspect of the invention is provided a method of treatment orprevention of an angiogenesis- and/or lymphangiogenesis-related disease,disorder or condition in a subject including the step of administeringto the subject an effective amount of the compound of the first aspect,or a pharmaceutically effective salt, solvate or prodrug thereof, or thepharmaceutical composition of the second aspect, to thereby treat orprevent the angiogenesis- and/or lymphangiogenesis-related disease,disorder or condition.

In a fourth aspect of the invention is provided use of the compound ofthe first aspect, or a pharmaceutically effective salt, solvate orprodrug thereof, in the manufacture of a medicament for the treatment orprevention of an angiogenesis- and/or lymphangiogenesis-related disease,disorder or condition.

In referring to the third and fourth aspects, the angiogenesis- and/orlymphangiogenesis-related disease, disorder or condition suitably is orcomprises an ophthalmic disease, disorder or condition. Preferably, theophthalmic disease, disorder or condition is selected from the groupconsisting of age-related macular degeneration, diabetic retinopathy,ischemic retinopathy, retinopathy of prematurity, neovascular glaucoma,iritis rubeosis, corneal neovascularization, cyclitis, sickle cellretinopathy, pterygium, vascular response during corneal injury and anycombination thereof.

In an alternative embodiment of the invention of the third and fourthaspects, the angiogenesis- and/or lymphangiogenesis-related disease,disorder or condition is or comprises a cancer. Preferably, the canceris selected from the group consisting of prostate cancer, lung cancer,breast cancer, bladder cancer, renal cancer, colon cancer, gastriccancer, pancreatic cancer, ovarian cancer, melanoma, hepatoma,hepatocellular carcinoma, sarcoma, leukemia, acute T cell lymphoma,vascular neoplasms and any combination thereof. In a particularembodiment, the compound of the first aspect or the pharmaceuticalcomposition of the second aspect prevents and/or inhibits metastasis ofsaid cancer.

In a further embodiment of the two aforementioned aspects, theangiogenesis- and/or lymphangiogenesis-related disease, disorder orcondition is or comprises a renal disease, disorder or condition.Preferably, the renal disease, disorder or condition is selected fromthe group consisting of chronic renal transplant dysfunction, primaryrenal fibrotic disorders, proteinuria, diabetic nephropathy, renalinflammation and any combination thereof.

In another embodiment of the two aforementioned aspects, theangiogenesis- and/or lymphangiogenesis-related disease, disorder orcondition is or comprises atherosclerosis.

In yet another embodiment of the two aforementioned aspects, theangiogenesis- and/or lymphangiogenesis-related disease, disorder orcondition is or comprises Hypotrichosis-Lymphedema-TelangiectasiaSyndrome.

In a fifth aspect of the invention is provided a method of inhibiting orpreventing metastasis of a cancer in a subject including the step ofadministering to the subject an effective amount of the compound of thefirst aspect, or a pharmaceutically effective salt, solvate or prodrugthereof, or the pharmaceutical composition of the second aspect, tothereby inhibit or prevent metastasis of the cancer.

Suitably, the cancer is selected from the group consisting of prostatecancer, lung cancer, breast cancer, bladder cancer, renal cancer, coloncancer, gastric cancer, pancreatic cancer, ovarian cancer, melanoma,hepatoma, sarcoma, leukemia, lymphoma, vascular neoplasms (e.g.,angioma, angiosarcoma, hemangioma) and any combination thereof.

In a sixth aspect of the invention is provided a method of inhibiting,preventing or reducing a SOX18 activity in a subject comprising the stepof administering to the subject an effective amount of the compound ofthe first aspect, or a pharmaceutically effective salt, solvate orprodrug thereof, or the pharmaceutical composition of the second aspect,to thereby inhibit, prevent or reduce the SOX18 activity in the subject.

Suitably, the SOX18 activity includes contacting and/or binding to a DNAsequence and/or a protein. Preferably, the protein is selected from thelist consisting of SOX7, RBPJ, XRCC5, SOX18, ILF3, DDX17 and anycombination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the invention may be readily understood and put intopractical effect, preferred embodiments will now be described by way ofexample with reference to the accompanying figures wherein:

FIG. 1 : Natural products, inhibitors of SOX18-DNA binding. A.Representative dataset from high throughput FP screen of 2688 marineextracts at 0.25 mg/mL. The screen was run on full-length mouse SOX18with FAM-labelled SOX-responsive element (mouse Prox1 intron 1).Competitive binding of a ligand to SOX18 reduces the FP index (arrowpointing to red dot active extract). B. Chemical structure of Sm1 andSm2. C. FP concentration-response curve of Sm1 and Sm2 (full-lengthmouse SOX18, mean±S.D. N=3).

FIG. 2 : Focused library of structural analogues and counter-screen within-silico aggregation predictor and critical micelle concentration (CMC)assay. A. First group is based on the ortho-hydroxybenzoic (salicylicacid) motif apparent in compounds Sm1 and Sm2. Second group is based ona similar resorcinol scaffold. Third group consists in approved NSAIDsthat contain a similar salicylic acid or anthranilic acid scaffold. B.Typical CMC data for neutral detergent Triton X100 control and twocompounds Sm4 and Sm10. C. SOX18-DNA binding inhibition by Sm4,meclofenamic, niflumic and flufenamic acids.

FIG. 3 : Compounds interact with SOX protein but not DNA. A.,B.Biotinylated double-strand DNA probes, approximately 40 base pair-long,with a SOX18 consensus element (A.) or a scrambled sequence (B.), andflanked with genomic DNA, were used to test small molecules DNA binding.Probes were immobilized on an SPR streptavidin chip. Positive controlsDAPI, ethidium bromide, and actinomycin D bind to DNA in a mannerconsistent with literature. Small molecule inhibitors (Sm4, 5 and 14) donot bind to consensus, or scrambled DNA. C. Thermostability ofSOX18[109] HMG fragment in the presence of either Prox1-DNA, Sm4, 5 orSm14, as measured by differential static light scattering of proteincomplex heated from 25 to 80° C. The binding of small molecules promotesprotein stability (ΔTagg>3° C. is considered a significantstabilization). Boltzmann curve fits of normalized light scatteringtriplicate data (Fitting goodness R2>0.97). D. Sm4 inhibits DNA bindingof SOX2, 6, 9, 11, 15, and 18-HMG fragments as measured with FP-basedDNA-binding competition assay.

FIG. 4 : Effect of Sm4, niflumic, flufenamic, and meclofenamic acids onSOX18 protein-protein interactions. A. Left Panel: Heatmap of SOX18pairwise protein-protein interactions as tested by ALPHAScreen, on XRCC6(negative control) and two proteins known to interact with SOX18, RBPJand MEF2C. Right Panel: Coimmunoprecipitation of protein complex.SOX18-mCherry-cMyc was co-expressed with either GFP-RBPJ, GFP-MEF2C, orGFP-only (negative control) under cell-free conditions andimmunoprecipitated with GFP Nanotrap beads. Bands: 1. RBPJ-GFP, 2.MEF2C-GFP, 3. SOX18-mCherry, 4. GFP. B. Effect of Sm4, niflumic,flufenamic and meclofenamic acids on SOX18 interaction with MEF2C andRBPJ.

FIG. 5 : Contribution of Sm4 chemical motives to SOX18 DNA bindinginhibition, SOX18-RBPJ binding inhibition, cytotoxicity and aggregation.Top-panel table depicts Sm14-44 compounds and summarizes in acolour-coded manner results obtained for four activity markers, namely,protein-DNA and protein-protein binding inhibition, cytotoxicity andaggregation risk. The second bottom bar graph details SOX18-RBPJprotein-protein binding inhibition results at 50 μM and 5 μM whereavailable (N=4, mean±SD). (DNA-binding inhibition, cytotoxicity andcLogP raw data are summarised in Table 3). The bottom bar graphillustrates SOX18-RBPJ protein-protein interaction (PPI) inhibition asmeasured by ALPHAScreen assay. Results are shown at 50 μM (left bars forPBS Ctrl, DMSO Ctrl and Sm4) and 5 μM (right bars for Sm4-Sm44). Of noteresults are shown when compound available (N=4, mean±SD). Comparison ofPPI disruption of the Sm4 series tested at 5 μM using SOX18 homodimerand SOX18/RBPJ heterodimer formation as a readout in ALPHAScreen assay.Some compounds preferentially disrupt SOX18 homodimer formation whereassome other are more specific to SOX18 heterodimer formation and someother are pan disruptors of both homo- and heterodimer complex.

FIG. 6 : In silico modelling of Sm4 at the interface between SOX18-HMGand RBPJ. Inhibition of SOX18-dependent transactivation in vitro. A.,B.Stable binding pose for Sm4 in the SOX18/Prox1 DNA X-ray crystalstructure, putting the inhibitor in an ‘open’ pocket between protein andDNA. C. Docking of the SOX18/DNA structure into the structure of theNotch transcription complex. D. Luciferase reporter assay in COS7 cellstransiently transfected with Sox18 and a vector containing Vcam1promoter merged to firefly luciferase gene (Hosking et al., 2004). Cellswere treated with small molecules at concentration below CC10 (10%cytotoxicity) for 24 hours in culture medium containing a maximum of 1%DMSO (v/v). Results are depicted for Sm4 and niflumic acid. Meclofenamicand flufenamic acids were inactive at concentrations below CC10; HumanSOX18-HMG region (Seq. ID No. 1) is shown; Mouse SOX18-HMG region (Seq.ID No. 2) is shown.

FIG. 7 : COX-1/2 enzyme inhibition. Inhibition of COX-1 and COX-2 enzymeby SOX18 inhibitors, including meclofenamic acid as positive control.Inhibition of COX enzyme is measured by the amount of PGH₂ prostanoidproduced from arachidonic acid conversion.

FIG. 8 : NMR spectra of Sm14-44.

FIG. 9 : Mapping of SOX18 interactome and disruption of interactions bySm4. (A) Schematic of the experimental strategy to deconvoluteSOX18-dependent protein-protein interactions (PPIs) combining Chromatinimmunoprecipitation-mass spectrometry (ChIP-MS) and AmplifiedLuminescent Proximity Homogeneous Assay (ALPHA-Screen) methods. (B)GO-term analysis for molecular function on the 289 proteins identifiedby SOX18-cMyc ChIP-MS in human umbilical vein endothelial cells(HUVECs). Non-specific interactors found in Myc-tag-only transfectedcells were subtracted. Proteins with nucleic acid binding or proteinbinding capacity (purple) were considered for consecutive directinteraction studies to enhance likeness of identifying directinteractors. (C) Left column: heatmap representation of SOX18 pairwisePPIs as tested by ALPHA-Screen, on a selection of ChIP-MS SOX18associated proteins, endothelial transcription factors andpositive/negative control proteins. Right column: heatmap representationof Sm4 activity on SOX18-dependent protein-protein interactions, astested at 100 μM. Interaction and disruption threshold is indicated inthe scale bar by a black line. Levels of interaction and disruptionabove the threshold are demarked by ‘+’, and below the threshold by ‘−’.Tagged proteins were expressed in the Leishmania tarentolae cell-freeprotein expression system. (D) Representative ALPHA-Screenconcentration-response curve for SOX18 PPI disruption by Sm4. Data shownare mean±s.e.m.

FIG. 10 : QC of SOX18 PPIs and effect of Sm4. (A) Mass spectrometryspectrum for a representative double charged DDX17 peptide with thesequence KAPILIATDVASRG (Muscat ion score 51.6), identified fromimmunoprecipitation of cMyc-SOX18 with anti-cMyc antibody in HUVECs. (B)Coverage of identified peptides of SOX18 and interacting proteinsselected from ChIP-MS. (C) Amino acid sequence of DDX17 (Seq. ID No. 3),with the identified ChIP-MS peptides indicated in green. (D) TypicalALPHA-Screen curve for protein dilution optimization, showing SOX9-SOX9and SOX18-SOX18. The presence of a peak (hook effect) demonstrates aninteraction and represents the ideal protein concentration forconsecutive binding studies. Proteins were expressed in the Leishmaniatarentolae cell-free protein expression system. (E) Molecular structureof SOX18 inhibitor Sm4. (F) ALPHA-Screen concentration-response curvesfor SOX18 PPI disruption by Sm4. Data shown are mean±s.e.m.

FIG. 11 : Differential disruption of SOXF PPI by Sm4. The left panelshows a matrix of protein-protein interactions between SOXF, MEF2C andRBPJ and OCT4 as measured by ALPHAScreen. The right panel shows theeffects of 50 uM Sm4 on PPIs (blue=no PPI/disruption, green/yellow=lowPPI/disruption, orange/red=strong PPI/complete disruption, grey=PPIbelow threshold, Sm4 effect cannot be determined).

FIG. 12 : Sm4 selectively affects SOX18 transcriptional output in vitro.(A) Schematic representation of the correlation analysis betweengenome-wide TF ChIP-seq data and Sm4 affected genes from transcriptomicsdata. The chromatin around the transcription start sites (TSS) of Sm4affected genes (purple) was investigated for transcription factorbinding peaks (grey), to calculate the “distance from TSS” to closestbinding site for a given transcription factor. This distance from TSSwas used as a proxy for the likelihood of transcriptional regulation,and thus make an association between Sm4 affected genes andtranscription factors (Cusanovich et al., PLoS Genetics, 2014; Verbistet al., Drug Discov Today, 2015). Included in the analysis where theChIP-seq peaks of SOX18 and SOX7, and of all transcription factorsavailable from the Encode consortium (GATA2, c-FOS, c-JUN, CTCF, EZH2,MAX and c-MYC), performed in HUVECs. A random group of genes wasanalysed as a control distribution as would be found by chance. (B) Sm4affected genes were grouped into down-regulated (Sm4-down), unaffected(Sm4-unchanged) and up-regulated (Sm4-up). The plots show the cumulativedistribution of the distance between the TSS of Sm4 affected genes(purple line, absolute fold change 2) and the closest genomic locationof binding sites for SOX18, and control transcription factors SOX7 andGATA2. The median distance from the TSS of differentially expressedgenes to the nearest binding event of a given transcription factor wascompared to the median distance that is expected by chance from a randomgene set (green line). Sm4 down regulated genes are significantly closer(bold) to the SOX18 peaks, but not to SOX7 or GATA2 peaks.

FIG. 13 : Transcriptome-wide analysis of Sm4 selectivity in vitro. (A)Top motif identified from SOX18 ChIP-seq peaks (MEME software) performedin HUVECs. (B) UCSC browser view of representative ChIP-seq peaks(arrowheads) for known SOX18 target genes VCAM and PROX1. (C) Conditionsfor transcriptome-wide analysis of Sm4. Differential expression (DE) wascalculated using DEseq2 in SOX18 overexpressing HUVECs, between vehicleDMSO (SOX18oe) and cells that received 25 μM Sm4 (Sm4) (D) Principalcomponent analysis of quadruplicate RNA-seq samples. Replicates samplesfrom same condition (control, SOX18oe, Sm4) cluster together. (E) Plotshowing a comparison between DESeq2 and edgeR methods, markingsignificance of DE genes between SOX18oe and Sm4 conditions. Transcriptswith a DESeq2 Log2 Fold Change 1 or −1 (dashed lines) were consideredfor further analysis. (F) The distance between Sm4 affected genes(purple) and the closest genomic location of binding sites a giventranscription factor, plotted as cumulative distribution. The mediandistance from the TSS of differentially expressed genes to the nearestbinding event of a transcription factor binding event was expressed as aratio over the median distance that is expected by chance (random genes,green).

FIG. 14 : c-JUN motifs are enriched in SOX18 binding sites. (A) HOMERmotif analysis on SOX18 ChIP-seq peaks revealed an enrichment of thec-JUN motif 5′-TGAC/GTCA-3′. (B) ALPHA-Screen binding curve forSOX18-c-JUN and SOX18-SOX18 (positive control), demonstrating that c-JUNhas the capacity to directly interact with SOX18 in vitro. Proteins wereexpressed in the Leishmania tarentolae cell-free protein expressionsystem.

FIG. 15 : Sm4 does not interfere with SOX9 or SOX17 activity in vitro.(A) Cell based reporter assay for SOX9 homodimer activity. COS-7 cellwere transfected with Sox9 and Col2a1:luc reporter construct. Sox9overexpression caused a >8-fold induction of Col2a1 activation. Nochange was observed at high concentration of Sm4. (B) Cell basedreporter assay for SOX17 activity (Robinson et al. 2014). Bovine AorticEndothelial Cells (BAECs) were transfected with pTK-β-gal (pTK) orECE1-TK-β-gal (ECE1) reporter, measuring endogenous activity of SOX17(ECE1-only). No change was observed at any of the tested concentration.Numbers on x-axis are [Sm4] in μM.

FIG. 16 : Sm4 blocks SoxF transcriptional activity in vivo. (A) Lateralbrightfield (top) and fluorescent (bottom) images of 60 hpf zebrafishlarvae carrying the tg(−6.5kdrl:eGFP) SoxF reporter. Treatment wasinitiated at late stage (20hpf) with either DMSO (negative control) or 1μM Sm4, or larvae were injected with morpholinos against both sox7 andsox18 (dMO sox7/18). Fluorescence intensity is shown as heatmap. Scalebar 200 μm (B) qRT-PCR analysis on gfp transcripts levels in treatedtg(−6.5kdrl:eGFP) larvae and sox7/18 morphants, showing reduction ofactivity on this transgene. (C) Lateral view of zebrafish larvaecarrying the tg(Dll4 in3:eGFP) SoxF/Notch reporter that harbors multiplebinding sites for Rbpj and SoxF transcription factors. Larvae wereinjected with a morpholino against rbpj and/or treated with 2 μM Sm4from 13 hpf. (D) qRT-PCR analysis on gfp transcripts in tg(Dll4in3:eGFP) larvae, showing repression of combined SoxF/Notch activity inthe Sm4-treated larvae. (E) Quantitation of embryonic lethality inlarvae, treated with Sm4 or DMSO control from early stage (16 hpf) until72 hpf. (F) Penetrance of vascular phenotype (arteriovenous shunting) in48 hpf larvae treated with 1.5 μM Sm4 from 16 hpf. (G) Penetrance ofcirculation defect in 48 hpf larvae treated with 1.5 μM Sm4 from 16 hpf.(H) qRT-PCR analysis of endogenous endothelial transcript levels at 48hpf in larvae treated with 1.5 μM Sm4 at 16 hpf, relative to DMSOcontrol (dotted line). Data shown are mean±s.e.m. *p<0.05, **p<0.01,***p<0.001.

FIG. 17 : Sox9 activity is not perturbed by treatment in vivo. (A)Timeline of treatment: Zebrafish larvae were treated continuously for 4days during chondrogenesis. Medium was refreshed daily throughout theexperiment to maintain Sm4 levels. (B) tg(col2a1:YFP) Sox9 reporterlarvae marking cartilage (Mitchell et al. 2013). YFP levels wereunaffected in presence of Sm4, and no changes in chondrogenesis wereobserved. mc: Meckel's cartilage, ch: ceratohyal, hs: hyosymplectic. (C)qRT-PCR of yfp transcript levels in DMSO control and Sm4 treated larvaeat a series of stages throughout chondrogenesis.

FIG. 18 : Sm4 interferes with SoxF activity in vivo. (A) Timeline of Sm4treatment in zebrafish larvae. Treatment for SOXF reporter gene studieswas initiated at 20 hpf, while for the phenotypic studies treatment wasinitiated at precedes that for, to act during the right developmentalwindow for arteriovenous specification. (B) Lateral view and transversesection of the trunk region of DMSO control and Sm4-treatedtg(fli1:eGFP,−6.5kdrl:mCherry) larvae. Control DMSO larvae formed adistinctly separated dorsal aorta (DA) and posterior cardinal vein(PCV). In Sm4-treated larvae, the DA was constricted and/or fused to thePCV (arrowheads). Whole mount in situ hybridization against arterialmarker efnb2a shows reduced expression and compromised formation of theDA and in Sm4-treated larvae at 48 hpf (arrows). Sections were DAPIstained (in blue). Scale bar brightfield: 0.5 mm, fluorescent and insitu 25 μm. (C) Concentration dependent effect of Sm4, showingquantitation for predominant phenotype at 72 hpf: mild (tail curvature),medium (dilation of the PCV) or severe (arteriovenous defect and/orcirculation defect). Indicated timeframe refers to Sm4 treatment windowand endpoint. (D) Quantitation of cardiac edema frequency in larvaetreated with Sm4 (1.5 μM). (E) qRT-PCR analysis of Sox18 dependent−6.5kdrl:mCherry and endogenous endothelial transcript levels inSm4-treated larvae relative to DMSO control (dotted line), showingeffect on arterial and venous markers at 24 hpf. All expression levelswere normalized to expression of endothelial marker cdh5. Data shown aremean±s.e.m. *p<0.05, **p<0.01, ***p<0.001.

FIG. 19 : Metastasis and tumor vascularization is suppressed by Sm4treatment. (A) Timeline of mouse model for breast cancer metastasis.4T1.2 tumor was inoculated at day 0, and resected at day 12. Sm4 (25mg/kg/day), Aspirin (25 mg/kg/day) or vehicle control (PBS), wasadministered orally on a daily basis from day 3 to day 12. Independentexperiments were carried out to assess survival and metastatic rate. (B)Blood plasma concentrations of Sm4 during the course of the treatmentscheme (day 7 and day 12) indicate good systemic delivery of the drug.(C) Expression of SOX18 in the vasculature of the tumor as shown by insitu hybridization. Scale bar 100 μm. (D) Survival of the mice wasmonitored (n=6-12 mice per group). Improved survival in Sm4-treated miceover both vehicle control and aspirin was analysed by Log-rank test(p<0.001). (E) No significant differences were found in tumor size atany stage. (F) Metastatic tumor nodules on the surface of the lungs werequantified at day 28, before any of the vehicle control or Sm4-treatedanimal had succumbed to the cancer burden. Data shown are mean±s.e.m of12-14 mice per group. (G) Vascular density was investigated on 300 μmsections of whole tumors. Bright field images show the overallmorphology of the tumor (outlined by dashed line) and presence of redblood cells, marking the main blood vessels and haemorrhagic areas(asterisks). Scale bar 1 mm. (H) Double immunofluorescence staining forendothelial specific markers ERG and Endomucin (EMCN) reveals thevascular patterning and penetration in the intra- and peri-tumoralregions. Left: whole tumor section (scale bar 1 mm), middle and right:blow-up of boxed regions (scale bar 200 μm). (I) Quantitation of EMCNvolume (blood vessel density) and ERG-positive nuclei (number ofendothelial cells) of n=6 tumours per condition. Each data pointrepresents the average of 3-4 representative regions (boxed areas inpanel H) per tumor. Mean±s.e.m for both conditions are shown. *p<0.05,**p<0.01.

FIG. 20 : Penetrance of blood vessels into 4T1.2 tumors is impaired bySm4. Brightfield images of serial vibratome sections (300 μm) from awhole 4T1.2 mammary tumor for mice treated with PBS vehicle or Sm4. Mainblood vessels and haemorrhagic areas are distinctive in red.

FIG. 21 : Sm4-treated mice have decreased tumor vascular density.Immunofluorescent staining for ERG and Endomucin (EMCN) on tumorsections. Two representative regions for both vehicle PBS and Sm4 areshown. Detailed blow-up shows distinct nuclear staining for ERG, andmembranous endothelial staining for EMCN. Quantitation of endothelialcells number and vascular volume was performed in Imaris on images withidentical XYZ dimensions. Thresholds were chosen to accurately capturetotal EMCN+vasculature and total ERG+ nuclei (ERG count and EMCN volumein yellow).

FIG. 22 : Sm4 treatment disrupts tumour-induced lymphangiogenesis.Lymphatic vessels images of serial vibratome sections (200 μm) from awhole 4T1.2 mammary tumor for mice treated with PBS vehicle or Sm4 (25mg/kg/day). Immunofluorescence for lymphatic specific markers PROX1 andPodoplanin (PDPN) and vascular EC marker Endomucin (EMCN) reveals thevascular patterning and penetration in the intra- and peri-tumoralregions. Whole tumor section for the control group (top panels), and forSm4 treated group (bottom panels). Quantitation of PDPN+ lymphaticvascular area (density, top graph) and PROX1+ nuclei (number oflymphatic endothelial cells, bottom graph) of n≥6 tumours per condition.Scale bar left: 0.5 mm, right: 0.1 mm. Mean±s.e.m for both conditionsare shown. **p<0.01, ***p<0.001.

FIG. 23 : (A) Principle for Single Molecule Tracking (SMT) experiment.SMT allows real-time imaging in live cells of chromatin binding dynamicsof transcription factors, such as SOX18. SMT determines a search patternof the SOX18 protein while it scans the genome to bind to specificresponsive elements of its target genes. (B) Panel B depicts theexperimental workflow, which involves bi-dimensional tracking ofmolecule trajectory and analysis using MATLAB. SOX18 molecules areeither bound to DNA (immobile) or unbound and freely diffusing in thenucleus. Within the immobile fraction it is possible to define 2populations either specific binding or non-specific binding based on thedwelling time on the chromatin. (C) Top; Sm4 increases SOX18 specificbound fraction at the expense of the non-specific bound fraction in aconcentration-dependent manner. Bottom; Sm4 increases the dwell time ofthe specifically bound SOX18 fraction while decreasing the dwell time ofthe non-specifically bound fraction. (D): Top; Sm4 selectively engageSOX18 dominant negative mutant Ra^(op) “Ragged Opossum” mutantcontributing to its partial rescuing as, conversely, it decreasesRa^(op) specific bound fraction at the benefit of the non-specific boundfraction, in a concentration-dependent manner. Bottom; Sm4 has the sameeffect on dwell times of specifically and non-specifically bound Ra^(op)fractions than on SOX18 fractions, but the effect is more marked.

DETAILED DESCRIPTION Definitions

In this patent specification, the terms ‘comprises’, ‘comprising’,‘includes’, ‘including’, or similar terms are intended to mean anon-exclusive inclusion, such that a method or composition thatcomprises a list of elements does not include those elements solely, butmay well include other elements not listed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as would be commonly understood by those ofordinary skill in the art to which this invention belongs.

The term “alkyl”, as used herein, refers to a straight-chain or branchedalkyl substituent containing from, for example, 1 to about 8 carbonatoms, preferably 1 to about 7 carbon atoms, more preferably 1 to about6 carbon atoms, even more preferably from 1 to about 4 carbon atoms.Examples of such substituents may be selected from the group consistingof methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl,tert-butyl, pentyl, isoamyl, 2-methylbutyl, 3-methylbutyl, hexyl,heptyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-ethylbutyl,3-ethylbutyl, octyl, nonyl, decyl, undecyl, dodecyl and the like. Thenumber of carbons referred to relates to the carbon backbone and carbonbranching but does not include carbon atoms belonging to anysubstituents, for example the carbon atoms of an alkyl or alkoxysubstituent branching off the main carbon chain.

The term “alkenyl” refers to optionally substituted unsaturated linearor branched hydrocarbon groups, having 2 to 8 carbon atoms, preferably 2to 7 carbon atoms, more preferably 2 to 6 carbon atoms or 2 to 4 carbonatoms and having at least one carbon-carbon double bond. Whereappropriate, the alkenyl group may have a specified number of carbonatoms, for example, C₂-C₆ alkenyl which includes alkenyl groups having2, 3, 4, 5 or 6 carbon atoms in linear or branched arrangements. Thenumber of carbons referred to relates to the carbon backbone and carbonbranching but does not include carbon atoms belonging to anysubstituents. Examples of such substituents may be selected from thegroup consisting of ethenyl, propenyl, isopropenyl, butenyl, s- andt-butenyl, pentenyl, hexenyl, hept-1,3-diene, hex-1,3-diene,non-1,3,5-triene and the like.

The term “alkoxyalkyl” as used herein means straight or branched chainalkyl groups linked by an oxygen atom (i.e., alkyl-O-alkyl otherwisereferred to as ‘ether’ groups), wherein alkyl is as described above. Inparticular embodiments, alkoxyalkyl refers to oxygen-linked groupscomprising 1 to 8 carbon atoms (“C1-8 alkoxyalkyl”). In furtherembodiments, alkoxyalkyl refers to oxygen-linked groups comprising 2 to8 carbon atoms (“C2-8 alkoxyalkyl”), 2 to 6 carbon atoms (“C2-6alkoxyalkyl”), 2 to 4 carbon atoms (“C2-4 alkoxyalkyl”) or 2 to 3 carbonatoms (“C2-3 alkoxyalkyl”). The recited number of carbon atoms refers tothose in the entire alkoxyalkyl/ether chain.

The term “optionally substituted”, as used herein, refers tosubstituents which may extend from the relevant group, such as a phenylor napthyl group, and may include such functionalities as halo includingF, Cl and Br; C₁-C₄ alkyl; OR₁₂ wherein R₁₂ is C₁-C₄ alkyl; and NR₁₃R₁₄wherein R₁₃ and R₁₄ are independently selected from H and C₁-C₄ alkyl.

According to a first aspect of the invention, there is provided acompound of formula (I), or a pharmaceutically acceptable salt, solvateor prodrug thereof:

wherein,

-   -   R₁ is selected from the group consisting of OH and OR₆ wherein        R₆ is C₁-C₄ alkyl;    -   R₂ is selected from the group consisting of H, COOR₇, and        C(O)NR₈R₉ wherein R₇, R₈ and R₉ are independently selected from        H and C₁-C₄ alkyl;    -   R₃ is L-A wherein L is a linker selected from C₂-C₈ alkyl, C₂-C₈        alkenyl and C₂-C₈ alkoxyalkyl and A is selected from optionally        substituted phenyl and optionally substituted napthyl;    -   R₄ is selected from the group consisting of H, OR₁₀, halo and        C₁-C₄ alkyl wherein R₁₀ is selected from H and C₁-C₄ alkyl; and    -   R₅ is selected from the group consisting of H, OR₁₁, halo and        C₁-C₄ alkyl wherein R₁₁ is selected from H and C₁-C₄ alkyl,    -   wherein, the compound is for use in the inhibition of a SOX18        activity.

In embodiments, R₁ is selected from the group consisting of OH and OMe.

Suitably, R₂ is selected from the group consisting of H, COOH, COOMe and

Preferably, R₂ is selected from COOH and

In embodiments, R₄ is selected from the group consisting of H, OH, OMe,Cl and Me.

Suitably, R₅ is selected from the group consisting of H, OH and OMe.

In certain embodiments, R₄ and R₅ are H.

In embodiments, L is a linker selected from C₂-C₆ alkyl, C₂-C₆ alkenyland C₂-C₆ alkoxyalkyl.

In any of the recited embodiments, R₃ is selected from the groupconsisting of:

wherein, the broken line indicates the attachment from that adjacentatom to the ring of formula I and the structures shown include E/Zisomers thereof.

In one embodiment, the compound of the first aspect is selected from thegroup consisting of:

In one preferred embodiment, the compound of the present aspect isselected from the group consisting of:

It would be understood by the skilled artisan that SOX18 is a member ofthe SOX (SRY-related HMG-box) family of transcription factors. Thesetranscription factors are typically involved in the regulation ofembryonic development and in the determination of the cell fate. Inparticular, the SOX18 protein can function as a transcriptionalregulator after forming a protein complex with other proteins. It hasbeen shown that SOX18 plays a role in hair, blood vessel, and lymphaticvessel development. Other names for SASH1 may include SRY-box 18, HLTSand HLTRS. Non-limiting examples of Accession Numbers referencing thenucleotide sequence of the SOX18 gene, or its encoded protein, as arewell understood in the art, in humans include NG_008095.1, NM_018419.2and NP_060889.1. As generally used herein, “SOX18” may refer to a SOX18nucleic acid or encoded protein, unless otherwise specified.

Suitably, the SOX18 activity that is modulated is that oflymphangiogenesis (i.e., the growth of new lymphatic vessels fromexisting lymphatic vessels), vasculogenesis (i.e., the de novo formationof the embryonic circulatory system) and/or angiogenesis (i.e., thegrowth of blood vessels from pre-existing vasculature). To this end, inan embodiment of the first aspect, one or more compounds of formula (I)may be useful for treating, decreasing or preventing lymphangiogenesis,angiogenesis and/or vasculogenesis.

Accordingly, one or more compounds of formula (I) suitably have aneffect in preventing and/or reducing the severity of the symptoms of anangiogensis- and/or lymphangiogenesis-related disease, disorder orcondition.

In one embodiment, the SOX18 activity includes contacting and/or bindingto a DNA sequence and/or a protein. In this regard, the compound of thefirst aspect may have an effect on one or more of the underlyingcellular signalling pathways of the angiogenesis- and/orlymphangiogenesis-related disease, disorder or condition, including, butnot limited to, the inhibition of SOX18 DNA binding and/orprotein-protein interactions.

With respect to DNA binding, it will be understood that SOX18 is atranscription factor capable of binding to DNA, such as to the consensussequence 5′-AACAAAG-3′ by its HMG box, so as to trans-activatetranscription via this binding. Furthermore, the SOX18 protein may actas a transcriptional regulator after forming a protein complex with oneor more proteins.

As used herein, a “gene” is a nucleic acid which is a structural,genetic unit of a genome that may include one or more aminoacid-encoding nucleotide sequences and one or more non-coding nucleotidesequences inclusive of promoters and other 5′ untranslated sequences,introns, polyadenylation sequences and other 3′ untranslated sequences,although without limitation thereto. In most cellular organisms, a geneis a nucleic acid that comprises double-stranded DNA.

The term “nucleic acid” as used herein designates single- ordouble-stranded DNA and RNA. DNA includes genomic DNA and cDNA. RNAincludes mRNA, RNA, RNAi, siRNA, cRNA and autocatalytic RNA. Nucleicacids may also be DNA-RNA hybrids. A nucleic acid comprises a nucleotidesequence which typically includes nucleotides that comprise an A, G, C,T or U base. However, nucleotide sequences may include other bases suchas inosine, methylycytosine, methylinosine, methyladenosine and/orthiouridine, although without limitation thereto.

By “protein” is meant an amino acid polymer. As would be appreciated bythe skilled person, the term “protein” also includes within its scopephosphorylated forms of a protein (i.e., a phosphoprotein) and/orglycosylated forms of a protein (i.e. a glycoprotein). A “peptide” is aprotein having no more than fifty (50) amino acids. A “polypeptide” is aprotein having more than fifty (50) amino acids.

Also provided are protein “variants” of SOX18 such as naturallyoccurring (e.g. allelic variants) and orthologs thereof. Preferably,protein variants share at least 70% or 75%, preferably at least 80% or85% or more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% sequence identity with an amino acid sequence of SOX18disclosed herein or known in the art.

As used herein, the term “protein-protein interaction” or “PPI” refersto refers to the close and stable association between two or moreproteins. It usually involves the formation of non-covalent chemicalbonds, such as hydrogen bonds. PPIs may be binary (two protein bindingpartners; a dimer) or tertiary (three or more protein binding partners,e.g., a trimer). Proteins within a PPI (i.e., binding partners) may bethe same protein (such as a homodimer or homotrimer) or differentproteins (such as a heterodimer or hetero trimer). Preferably, theprotein interaction is reversible such that dissociation of SOX18 fromthe protein, or protein subunits, can occur under suitable conditions.Preferably, such forces are weak, e.g. have K_(d)'s in the μM range,such that the compound of the invention can disrupt the interactionbetween the SOX18 and the protein.

Preferably, the protein is selected from the list consisting of SOX7,RBPJ, XRCC5, SOX18, ILF3, DDX17 and any combination thereof.

In view of the above, it is one advantage of the compounds of the firstaspect that, depending on the selection of groups around the core phenylring, the clinical effect they display may be somewhat tailored,depending on the choice of groups, towards the inhibition of DNA bindingand/or particular protein-protein interactions by SOX18.

In some embodiments, compounds with one or more chiral centers may beprovided. While racemic mixtures of compounds of the invention may beactive, selective, and bioavailable, isolated isomers may be of interestas well.

The compounds of the present invention also include stereoisomers of thecompounds described herein and compositions comprising more than onecompound of the invention may, where applicable, include suchstereoisomers, for example E/Z isomers, either individually or admixedin any proportions. Stereoisomers may include, but are not limited to,enantiomers, diastereomers, racemic mixtures, and combinations thereof.Such stereoisomers can be prepared and separated using conventionaltechniques, either by reacting enantiomeric starting materials, or byseparating isomers of compounds and prodrugs of the present invention.Isomers may include geometric isomers. Examples of geometric isomersinclude, but are not limited to, trans isomers or cis isomers (E/Z)across a double bond. Other isomers are contemplated among the compoundsof the present invention. The isomers may be used either in pure form orin admixture with other isomers of the compounds described herein.

Various methods are known in the art for preparing optically activeforms and determining activity. Such methods include standard testsdescribed herein and other similar tests which are well known in theart. Examples of methods that can be used to obtain optical isomers ofthe compounds according to the present invention include the following:

-   -   i) physical separation of crystals whereby macroscopic crystals        of the individual enantiomers are manually separated. This        technique may particularly be used when crystals of the separate        enantiomers exist (i.e., the material is a conglomerate), and        the crystals are visually distinct;    -   ii) simultaneous crystallization whereby the individual        enantiomers are separately crystallized from a solution of the        racemic, possible only if the latter is a conglomerate in the        solid state;    -   iii) enzymatic resolutions whereby partial or complete        separation of a racemate by virtue of differing rates of        reaction for the enantiomers with an enzyme;    -   iv) enzymatic asymmetric synthesis, a synthetic technique        whereby at least one step of the synthesis uses an enzymatic        reaction to obtain an enantiomerically pure or enriched        synthetic precursor of the desired enantiomer;    -   v) chemical asymmetric synthesis whereby the desired enantiomer        is synthesized from an achiral precursor under conditions that        produce asymmetry (i.e., chirality) in the product, which may be        achieved using chiral catalysts or chiral auxiliaries;    -   vi) diastereomer separations whereby a racemic compound is        reacted with an enantiomerically pure reagent (the chiral        auxiliary) that converts the individual enantiomers to        diastereomers. The resulting diastereomers are then separated by        chromatography or crystallization by virtue of their now more        distinct structural differences and the chiral auxiliary later        removed to obtain the desired enantiomer;    -   vii) first- and second-order asymmetric transformations whereby        diastereomers from the racemate equilibrate to yield a        preponderance in solution of the diastereomer from the desired        enantiomer or where preferential crystallization of the        diastereomer from the desired enantiomer perturbs the        equilibrium such that eventually in principle all the material        is converted to the crystalline diastereomer from the desired        enantiomer. The desired enantiomer is then released from the        diastereomers;    -   viii) kinetic resolutions comprising partial or complete        resolution of a racemate (or of a further resolution of a        partially resolved compound) by virtue of unequal reaction rates        of the enantiomers with a chiral, non-racemic reagent or        catalyst under kinetic conditions;    -   ix) enantiospecific synthesis from non-racemic precursors        whereby the desired enantiomer is obtained from non-chiral        starting materials and where the stereochemical integrity is not        or is only minimally compromised over the course of the        synthesis;    -   x) chiral liquid chromatography whereby the enantiomers of a        racemate are separated in a liquid mobile phase by virtue of        their differing interactions with a stationary phase. The        stationary phase can be made of chiral material or the mobile        phase can contain an additional chiral material to provoke the        differing interactions;    -   xi) chiral gas chromatography whereby the racemate is        volatilized and enantiomers are separated by virtue of their        differing interactions in the gaseous mobile phase with a column        containing a fixed non-racemic chiral adsorbent phase;    -   xii) extraction with chiral solvents whereby the enantiomers are        separated by virtue of preferential dissolution of one        enantiomer into a particular chiral solvent; and    -   xiii) transport across chiral membranes whereby a racemate is        placed in contact with a thin membrane barrier. The barrier        typically separates two miscible fluids, one containing the        racemate, and a driving force such as concentration or pressure        differential causes preferential transport across the membrane        barrier. Separation occurs as a result of the non-racemic chiral        nature of the membrane which allows only one enantiomer of the        racemate to pass through.

The compound of the first aspect may optionally be provided in acomposition that is enantiomerically or diastereomerically enriched,such as a mixture of enantiomers or diastereomers in which oneenantiomer or diastereomer is present in excess, in particular, to theextent of 95% or more, 96% or more, 97% or more, 98% or more, or 99% ormore, including 100%.

The compounds of the first aspect may be utilized per se or in the formof a pharmaceutically acceptable ester, amide, salt, solvate, prodrug,or isomer, as appropriate. For example, the compound may be provided asa pharmaceutically acceptable salt. If used, a salt of the drug compoundshould be both pharmacologically and pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare the free active compound or pharmaceutically acceptable saltsthereof and are not excluded from the scope of this invention. Suchpharmacologically and pharmaceutically acceptable salts can be preparedby reaction of the drug with an organic or inorganic acid, usingstandard methods detailed in the literature.

Examples of pharmaceutically acceptable salts of the compounds usefulaccording to the invention include acid addition salts. Salts ofnon-pharmaceutically acceptable acids, however, may be useful, forexample, in the preparation and purification of the compounds. Suitableacid addition salts according to the present invention include organicand inorganic acids. Preferred salts include those formed fromhydrochloric, hydrobromic, sulfuric, phosphoric, citric, tartaric,lactic, pyruvic, acetic, succinic, fumaric, maleic, oxaloacetic,methanesulfonic, ethanesulfonic, p-toluenesulfonic, benzenesulfonic, andisethionic acids. Other useful acid addition salts include propionicacid, glycolic acid, oxalic acid, malic acid, malonic acid, benzoicacid, cinnamic acid, mandelic acid, salicylic acid, and the like.Particular example of pharmaceutically acceptable salts include, but arenot limited to, sulfates, pyrosulfates, bisulfates, sulfites,bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates,metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates,propionates, decanoates, caprylates, acrylates, formates, isobutyrates,caproates, heptanoates, propiolates, oxalates, malonates, succinates,suberates, sebacates, fumarates, maleates, butyne-1,4-dioates,hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates,dinitrobenzoates, hydroxybenzoates, methoxyenzoates, phthalates,sulfonates, xylenesulfonates, phenylacetates, phenylpropionates,phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolates,tartrates, methanesulfonates, propanesulfonates,naphthalene-1-sulfonates, naphthalene-2-sulfonates, and mandelates.

An acid addition salt may be reconverted to the free base by treatmentwith a suitable base. Preparation of basic salts of acid moieties whichmay be present on a compound or prodrug useful according to the presentinvention may be prepared in a similar manner using a pharmaceuticallyacceptable base, such as sodium hydroxide, potassium hydroxide, ammoniumhydroxide, calcium hydroxide, triethylamine, or the like.

Esters of the compounds according to the present invention may beprepared through functionalization of hydroxyl and/or carboxyl groupsthat may be present within the compound. Amides and prodrugs may also beprepared using techniques known to those skilled in the art. Forexample, amides may be prepared from esters, using suitable aminereactants, or they may be prepared from an anhydride or an acid chlorideby reaction with ammonia or a lower alkyl amine. Moreover, esters andamides of compounds of the invention can be made by reaction with acarbonylating agent (e.g., ethyl formate, acetic anhydride,methoxyacetyl chloride, benzoyl chloride, methyl isocyanate, ethylchloroformate, methanesulfonyl chloride) and a suitable base (e.g.,4-dimethylaminopyridine, pyridine, triethylamine, potassium carbonate)in a suitable organic solvent (e.g., tetrahydrofuran, acetone, methanol,pyridine, N,N-dimethylformamide) at a temperature of 0° C. to 60° C.

Examples of pharmaceutically acceptable solvates include, but are notlimited to, compounds according to the invention in combination withwater, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid,or ethanolamine.

According to a second aspect of the invention there is provided apharmaceutical composition comprising a compound of the first aspect, ora pharmaceutically acceptable salt, solvate or prodrug thereof, and apharmaceutically acceptable carrier, diluent and/or excipient.

Suitably, the pharmaceutically acceptable carrier, diluent and/orexcipient may be or include one or more of diluents, solvents, pHbuffers, binders, fillers, emulsifiers, disintegrants, polymers,lubricants, oils, fats, waxes, coatings, viscosity-modifying agents,glidants and the like.

The salt forms of the compounds of the invention may be especiallyuseful due to improved solubility.

Diluents may include one or more of microcrystalline cellulose, lactose;mannitol, calcium phosphate, calcium sulfate; kaolin, dry starch,powdered sugar, and the Ike. Binders may include one or more ofpovidone, starch, stearic acid, gums, hydroxypropylmethyl cellulose andthe like. Disintegrants may include one or more of starch,croscarmellose sodium, crospovidone, sodium starch glycolate and theIke, Solvents may include one or more of ethanol, methanol, isopropanol,chloroform, acetone, methylethyl ketone, methylene chloride, water andthe like. Lubricants may include one or more of magnesium stearate, zincstearate, calcium stearate, stearic acid, sodium stearyl fumarate,hydrogenated vegetable oil, glyceryl behenate and the like. A glidantmay be one or more of colloidal silicon dioxide, talc or cornstarch andthe like. Buffers may include phosphate buffers, borate buffers andcarbonate buffers, although without limitation thereto. Fillers mayinclude one or more gels inclusive of gelatin, starch and syntheticpolymer gels, although without limitation thereto. Coatings may compriseone or more of film formers, solvents, plasticizers and the Ike.Suitable film formers may be one or more of hydroxypropyl methylcellulose, methyl hydroxyethyl cellulose, ethyl cellulose, hydroxypropylcellulose, povidone, sodium carboxymethyl cellulose, polyethyleneglycol, acrylates and the Ike. Suitable solvents may be one or more ofwater, ethanol, methanol, isopropanol, chloroform, acetone, methylethylketone, methylene chloride and the Ike. Plasticizers may be one or moreof propylene glycol, castor oil, glycerin, polyethylene glycol,polysorbates, and the like.

Reference is made to the Handbook of Excipients 6th Edition, Eds. Rowe,Sheskey & Quinn (Pharmaceutical Press), which provides non-limitingexamples of excipients which may be useful according to the invention.

It will be appreciated that the choice of pharmaceutically acceptablecarriers, diluents and/or excipients will, at least in part, bedependent upon the mode of administration of the formulation. By way ofexample only, the composition may be in the form of a tablet, capsule,caplet, powder, an injectable liquid, a suppository, a slow releaseformulation, an osmotic pump formulation or any other form that iseffective and safe for administration.

Suitably, the pharmaceutical composition is for the treatment orprevention of a disease, disorder or condition in a mammal as describedbelow. Preferably, the pharmaceutical composition is for the treatmentor prevention of an angiogenesis- and/or lymphangiogenesis-relateddisease, disorder or condition in a mammal.

A third aspect of the invention resides in a method of treatment orprevention of an angiogenesis- and/or lymphangiogenesis-related disease,disorder or condition including the step of administering an effectiveamount of a compound of the first aspect, or a pharmaceuticallyeffective salt, solvate or prodrug thereof, or the pharmaceuticalcomposition of the second aspect, to thereby treat or prevent theangiogenesis- and/or lymphangiogenesis-related disease, disorder orcondition.

A fourth aspect of the invention provides for use of a compound of thefirst aspect, or a pharmaceutically effective salt, solvate or prodrugthereof, in the manufacture of a medicament for the treatment orprevention of a disease, disorder or condition, such as an angiogenesis-and/or lymphangiogenesis-related disease, disorder or condition.

As generally used herein, the terms “administering” or “administration”,and the like, describe the introduction of the compound or compositionto a mammal such as by a particular route or vehicle. Routes ofadministration may include topical, parenteral and enteral which includeoral, buccal, sub-lingual, nasal, anal, gastrointestinal, subcutaneous,intramuscular and intradermal routes of administration, although withoutlimitation thereto.

As used herein, “treating” (or “treat” or “treatment”) refers to atherapeutic intervention that ameliorates a sign or symptom of theangiogenesis- and/or lymphangiogenesis-related disease, disorder orcondition after it has begun to develop. The term “ameliorating”, withreference to the angiogenesis- and/or lymphangiogenesis-related disease,disorder or condition, refers to any observable beneficial effect of thetreatment. Treatment need not be absolute to be beneficial to thesubject. The beneficial effect can be determined using any methods orstandards known to the ordinarily skilled artisan.

As used herein, “preventing” (or “prevent” or “prevention”) refers to acourse of action (such as administering a therapeutically effectiveamount of one or more of the compounds described herein) initiated priorto the onset of a symptom, aspect, or characteristic of theangiogenesis- and/or lymphangiogenesis-related disease, disorder orcondition so as to prevent or reduce the symptom, aspect, orcharacteristic. It is to be understood that such preventing need not beabsolute to be beneficial to a subject. A “prophylactic” treatment is atreatment administered to a subject who does not exhibit signs of anangiogenesis- and/or lymphangiogenesis-related disease, disorder orcondition or exhibits only early signs for the purpose of decreasing therisk of developing a symptom, aspect, or characteristic of theangiogenesis- and/or lymphangiogenesis-related disease, disorder orcondition.

As used herein, “effective amount” refers to the administration of anamount of the relevant compound or composition sufficient to prevent theoccurrence of symptoms of the condition being treated, or to bring abouta halt in the worsening of symptoms or to treat and alleviate or atleast reduce the severity of the symptoms. The effective amount willvary in a manner which would be understood by a person of skill in theart with patient age, sex, weight etc. An appropriate dosage or dosageregime can be ascertained through routine trial.

As used herein, the terms “subject” or “individual” or “patient” mayrefer to any subject, particularly a vertebrate subject, and even moreparticularly a mammalian subject, for whom therapy is desired. Suitablevertebrate animals include, but are not restricted to, primates, avians,livestock animals (e.g., sheep, cows, horses, donkeys, pigs), laboratorytest animals (e.g., rabbits, mice, rats, guinea pigs, hamsters),companion animals (e.g., cats, dogs) and captive wild animals (e.g.,foxes, deer, dingoes). A preferred subject is a human in need oftreatment for an angiogenesis- and/or lymphangiogenesis-related disease,disorder or condition, as described herein. However, it will beunderstood that the aforementioned terms do not imply that symptoms arenecessarily present.

The term “angiogenesis-related disease, disorder or condition” as usedherein denotes any disorder associated with abnormal blood vesselgrowth, including excessive blood vessel growth. It will be understoodthat the control of angiogenesis is altered in certain diseases,disorders or conditions. Many such diseases involve pathologicalangiogenesis (i.e., inappropriate, excessive or undesired blood vesselformation), which supports the disease state and, in many instances,contributes to the cellular and tissue damage associated with suchdiseases. Angiogenesis-related diseases, disorder or conditions (i.e.,those involving pathological angiogenesis) can be many and varied, andmay include, for example, various types of cancers, chronic inflammatorydiseases, and neovascularization diseases. Examples of chronicinflammatory diseases, disorders or conditions include, but are notlimited to, inflammatory bowel disease, such as Crohn's disease andulcerative colitis, rheumatoid arthritis, lupus, psoriasis,atherosclerosis and diabetes mellitus.

As generally used herein, the term “lymphangiogenesis-related disease,disorder or condition” refers to any disorder associated with abnormallymphatic vessel growth, including excessive lymphatic vessel growth.Lymphangiogenesis is ultimately controlled by a complex network ofgrowth factors, cytokines and chemokines and can occur under a number ofpathological conditions (see, e.g., El-Chemaly, Ann N Y Acad Sci (2008);Patel, Seminars Opthalmol (2009); El-Chemaly, Lymphatic Res Biol (2009);Pepper, Clin Cancer Res (2001)), including, but not limited to cancergrowth and metastasis, inflammation and transplant rejection. Withrespect to metastasis, cancer cells may metastasize to lymph nodes anddistal organs through lymphatic vessels and this often represents thefirst step in cancer cell spread beyond the primary cancer.

In one embodiment of the third and fourth aspects, the angiogenesis-and/or lymphangiogenesis-related disease, disorder or condition is orcomprises an ophthalmic disease, disorder or condition, and inparticular those involving neovascularization. To this end, angiogenesisand/or lymphangiogenesis can play a pivotal role in the development ofophthalmic diseases, disorders or conditions, such as age-relatedmacular degeneration, diabetic retinopathy, ischemic retinopathy,retinopathy of prematurity, neovascular glaucoma, iritis rubeosis,corneal neovascularization, cyclitis, sickle cell retinopathy, thevascular response during corneal injury and pterygium. As theseophthalmic diseases, disorders or conditions progress, the blood vesselsof the eye may not only proliferate excessively, but the new vessels canalso be weak, leaky and prone to hemorrhage. To this end, the newabnormal vessels may bleed and cause subsequent blindness in thesubject.

In one embodiment of the third and fourth aspects, the angiogenesis-and/or lymphangiogenesis-related disease, disorder or condition is orcomprises a cancer. To this end, the formation and metastasis of acancer typically involves pathological angiogenesis. Similar to healthytissues, cancers require new blood vessel formation in order to providenutrients and oxygen and remove cellular wastes. Thus, pathologicalangiogenesis is critical to the growth and expansion of a cancer.

As generally used herein, the terms “cancer”, “tumour”, “malignant” and“malignancy” refer to diseases or conditions, or to cells or tissuesassociated with the diseases or conditions, characterized by aberrant orabnormal cell proliferation, differentiation and/or migration oftenaccompanied by an aberrant or abnormal molecular phenotype that includesone or more genetic mutations or other genetic changes associated withoncogenesis, expression of tumour markers, loss of tumour suppressorexpression or activity and/or aberrant or abnormal cell surface markerexpression.

Cancer may include any aggressive or potentially aggressive cancers,tumours or other malignancies such as listed in the NCI Cancer Index athttp://www.cancer.gov/cancertopics/alphalist, including all major cancerforms such as sarcomas, carcinomas, lymphomas, leukaemias and blastomas,although without limitation thereto. These may include breast cancer,lung cancer inclusive of lung adenocarcinoma, cancers of thereproductive system inclusive of ovarian cancer, cervical cancer,uterine cancer and prostate cancer, cancers of the brain and nervoussystem, head and neck cancers, gastrointestinal cancers inclusive ofcolon cancer, colorectal cancer and gastric cancer, liver cancer, kidneycancer, skin cancers such as melanoma and skin carcinomas, blood cellcancers inclusive of lymphoid cancers and myelomonocytic cancers,cancers of the endocrine system such as pancreatic cancer and pituitarycancers, musculoskeletal cancers inclusive of bone and soft tissuecancers, vascular cancers or neoplasms, such as hemangioma, angioma andangiosarcoma, although without limitation thereto.

In particular embodiments, the cancer is selected from the groupconsisting of prostate cancer, lung cancer, breast cancer, bladdercancer, renal cancer, colon cancer, gastric cancer, pancreatic cancer,ovarian cancer, melanoma, hepatoma, hepatocellular carcinoma, sarcoma,leukemia, lymphoma, vascular neoplasms, such as hemangioma, angioma andangiosarcoma and any combination thereof.

In one particular embodiment, the compound of the first aspect or thepharmaceutical composition of the second aspect prevents and/or inhibitsmetastasis of said cancer.

As used herein, “metastasis” or “metastatic”, refers to the migration ortransfer of malignant cancer cells, or neoplasms, via the circulatory orlymphatic systems or via natural body cavities, typically from theprimary focus of tumour, cancer or a neoplasia to a distant site in thebody, and the subsequent development of one or more secondary tumours orcolonies thereof in the one or more new locations. “Metastases” refersto the secondary tumours or colonies formed as a result of metastasisand encompasses micro-metastases as well as regional and distantmetastases.

It will be appreciated that pathological angiogenesis andlymphangiogenesis may play an important role in cancer metastasis. Tothis end, the formation of blood vessels in a primary cancer not onlyallows cancer cells to enter the blood stream and to circulatethroughout the body, but also supports the formation and growth ofmetastatic cancers seeded by cancer cells that have metastasized fromthe primary site.

In a further embodiment of the two aforementioned aspects, theangiogenesis- and/or lymphangiogenesis-related disease, disorder orcondition is or comprises a renal disease, disorder or condition.Preferably, the renal disease, disorder or condition is selected fromthe group consisting of chronic renal transplant dysfunction, primaryrenal fibrotic disorders, proteinuria, diabetic nephropathy, renalinflammation and any combination thereof.

In one particular embodiment of the third and fourth aspects, theangiogenesis- and/or lymphangiogenesis-related disease, disorder orcondition is or comprises atherosclerosis. To this end, it would beappreciated by the skilled artisan that the pathological changes inatherosclerosis can at least in part be attributed to chronicinflammation and neovascularisation. Furthermore, a link has beendemonstrated between NF-kB-dependent atherogenic inflammatory response,and SOX18 regulation, suggesting that SOX18 may play a role in thedevelopment of atherosclerosis (Garcia-Ramirez et al., 2005). Sox18 hasalso been shown to be overexpressed in atherosclerotic plaques, andhence could be a major component of the disease aetiology (Brown et al.,2014).

In one embodiment of the third and fourth aspects, the angiogenesis-and/or lymphangiogenesis-related disease, disorder or condition is orcomprises Hypotrichosis-Lymphedema-Telangiectasia Syndrome. In thisregard, it would be appreciated thatHypotrichosis-Lymphedema-Telangiectasia Syndrome is associated withmutations in the SOX18 gene.

In a fifth aspect, the invention provides a method of preventing orinhibiting metastasis of a cancer in a subject including the step ofadministering to the subject an effective amount of the compound of thefirst aspect, or a pharmaceutically effective salt, solvate or prodrugthereof, or the pharmaceutical composition of the second aspect, tothereby inhibit or prevent metastasis of the cancer.

Suitably, the cancer is that hereinbefore described.

A sixth aspect of the invention resides in a method of inhibiting,preventing or reducing a SOX18 activity in a subject comprising the stepof administering an effective amount of a compound of the first aspect,or a pharmaceutically effective salt, solvate or prodrug thereof, or thepharmaceutical composition of the second aspect, to thereby inhibit,prevent or reduce the SOX18 activity in the subject.

Suitably, the SOX18 activity includes contacting and/or binding to a DNAsequence and/or a protein. Preferably, the protein is selected from thelist consisting of SOX7, RBPJ, XRCC5, SOX18, ILF3, DDX17 and anycombination thereof.

The various features and embodiments of the present invention, referredto in individual sections above apply, as appropriate, to othersections, mutatis mutandis. Consequently features specified in onesection may be combined with features specified in other sections asappropriate.

The following experimental section describes in more detail thecharacterisation of certain of the compounds of the invention and theirefficacy. The intention is to illustrate certain specific embodiments ofthe compounds of the invention and their efficacy without limiting theinvention in any way.

Example 1 Materials and Methods Compounds Preparation

Marine extract library. A library of n-butanol fractions generated froma marine library collected across Australia and Antarctica was used forscreening. Active fractions were fractionated into pure compoundsre-assayed in the same way as original fractions.

A library of 2688 samples of marine invertebrate and alga collectedacross southern Australia and Antarctica was processed to generate anextract library suitable for high throughput bioassay. EtOH extractswere decanted, concentrated, and partitioned into n-BuOH and H₂O phases,then transferred to deep 96-well plates, resulting in a >10-foldconcentration of small molecules, while removing salts. The n-BuOHfraction (25 mg/mL w/v of dried residue) was used for screening,following 10- and 100-fold dilution (2.5 and 0.25 mg/mL). Activefractions were triturated with hexane, CH2Cl2 and MeOH, and fractionatedinto pure compounds by HPLC. All compounds were assayed in the same wayas fractions.

Sm1 (6-undecyl salicylic acid) and Sm2 (6-tridecyl salicylic acid) werepurified from the original aqueous EtOH extract of brown alga sample,Caulocystis cephalornithos (CMB-01671). The aqueous EtOH extract ofsample of brown alga, Caulocystis cephalornithos (CMB-01671), wasconcentrated in vacuo (4.8 g) and partitioned into n-BuOH (0.80 g) andH₂O (4.0 g) soluble materials. An aliquot (40 mg) of the n-BuOH solublepartition was subjected to HPLC fractionation (Agilent Zorbax SB C18 5μm, 250×9.4 mm column, 4 mL/min gradient elution from 60% MeOH/H2O to100% MeOH over 10 min, followed with 100% MeOH for 10 min, withisocratic 0.01% TFA modifier) to yield 6-undecyl salicylic acid (Sm1,5.9 mg, RT 12.8 min) and 6-tridecyl salicylic acid (Sm2, 11 mg, RT 13.7min).

Sm4-Sm14—Focused library. The synthetic analogues were purchased fromEndoTherm GmbH (Germany) and Princeton BioMolecular Research (USA), andanalyzed for purity by HP-LC/MS.

Sm15-Sm44 SAR library. The SAR library was designed to investigate therole of the lipophilic tail and possible substituents of the salicylicacid scaffold. The six-step synthesis outlined below started from asubstituted benzoic acid using a Wittig olefination reaction tointroduce the lipophlic tail. Partially protected intermediates werealso included in the SAR library. All compounds were purified by HPLC(purity >90%, UV/ELSD/MS).

General Materials and Methods. Reagents and anhydrous solvents (THF,dichloromethane, and acetonitrile) were used as received. Reactionprogress was monitored by TLC using Merck silica gel 60 F-254 with UVdetection. Silica gel 60 (Merck 40-63 pm) was used for columnchromatography. The following stain solutions have been used in additionto UV light with fluorescent TLC plates: phosphomolybdic acid,anisaldehyde/EtOH. Reactions requiring anhydrous conditions wereperformed under nitrogen. NMR data were collected and calibrated ind4-MeOH or CDCl3 at 298K on a Varian Unity 400 MHz or Bruker Avance 600MHz spectrometers. HPLC and routine mass spectra were acquired on anAgilent Technologies 1200 Series instrument, fitted with a G1316A UV-Visdetector, 1200 Series ELSD and 6110 quadrupole ESI-MS. High resolutionmass spectrometry (HRMS) was performed on the Bruker MicroTOF massspectrometer.

General procedure for preparation of N,N-diethylbenzamides (4a-d)1. Thesubstituted N,N-diethyl benzamides were prepared from the respectivesubstituted benzoic acids. The acid (5 g, 32.9 mmol) was refluxed withexcess thionyl chloride (50 mL) until the evolution of hydrogen chloridewas ceased. The excess thionyl chloride was removed under reducedpressure and co-distilled with toluene (3×15 mL). The acid chloride wasdissolved in dry CH₂Cl2 (100 mL) and added drop wise diethylamine (13.6mL, 131.5 mmol) at 0° C. and stirred at room temperature for overnight.The reaction mixture was diluted with CH₂Cl₂ (100 mL), washed with water(50 mL), brine solution and dried over anhydrous MgSO₄. The organiclayer was removed on rotovapor to give the crude compound. The crudecompound was purified by flash column chromatography to obtain the purediethylbenzamide.

General procedure for directed ortho-metalation reaction (5a-d)2. To asolution of TMEDA (0.55 mL, 3.7 mmol) in dry THF (10 mL) was addeds-BuLi (2.6 mL, 3.7 mmol, 1.5M in cyclohexane) at −78° C. and stirredfor 15 min., followed by 1-methoxy-N,N-diethylbenzamide (0.35 g, 1.7mmol) in THF (5 mL). After stirring at the same temperature for 2 h,anhydrous DMF (0.52 mL, 6.8 mmol) was added slowly. The reaction mixturewas gradually warmed up to room temperature and stirred for 30 min. Thereaction was quenched by addition of 6N aq. HCl solution (10 mL), andextracted with ethyl acetate (3×15 mL). The combined organic layers werewashed with brine (10 mL), and dried over MgSO4. After removal of thesolvent under vacuum, the residue was purified by a flash columnchromatography (n-hexane/ethyl acetate, 1/2) to give the product.

General procedure for cleaving N,N-diethylbenzamide (10a-d)3N,N-diethylbenzamide (1.3 mmol) was dissolved in glacial AcOH (3 mL),added 10% aq. HCl (3 mL), and the mixture was refluxed for 12 h. Aftercooling to room temperature, the acetic acid was removed under reducedpressure, diluted with H₂O and extracted with EtOAc (30 mL). The organiclayer was washed with brine, separated and dried over anhydrous MgSO4.The solvent was removed under reduced pressure to give the product.

General procedure for Wittig olefination reaction (12a-d)4. To asuspension of Wittig salt (1.0 mmol) in THF (10 mL) was added t-BuOK(2.0 mmol) or NaH (2.0 mmol) at 0° C. and stirred for 30 min. Thealdehyde (0.8 mmol) dissolved in THF was added slowly and stirred forovernight at room temperature (at 50° C. for reactions carried usingNaH, entries 2, 4, 5, 6). The reaction was quenched with H₂O andextracted into EtOAc (30 mL). The organic layer was washed brine (20mL), dried over MgSO4, filtered, and concentrated in vacuo to give thecrude compound. The crude compound was purified by flash columnchromatography to obtain pure product.

General procedure for demethylation: Method A (using BBr3)5. A solutionof compound (74 mg, 0.192 mmol) in CH2Cl2 (70 mL) at −78° C. was treatedwith BBr3 (1.0 M in CH2Cl2, 0.576 mL, 0.576 mmol). The mixture wasstirred at −78° C. for 2 h, and then quenched with saturated aq. NH4Cl(10 mL). The mixture was allowed to warm up to room temperature anddiluted with CH2Cl2 (30 mL). The organic layer was washed with brine (10mL), dried over MgSO4, filtered, and concentrated in vacuo.

General procedure for the demethylation: Method B (using HBr)6. Thesolution of compound (100 mg, 0.192 mmol) in 48% aq. HBr (3.0 mL) washeated to reflux for 3 h. After completion of the reaction, was allowedto warm to room temperature and evaporated under reduced pressure togive crude residue. The crude compound was extracted with EtOAc (30 mL)and washed with H₂O (20 mL). The organic layer was separated, dried(MgSO4) and concentrated to give the product.

General method for reduction of olefin (14a-d). To a solution of olefinin 1:1 (EtOAc:MeOH) was added 10% Pd/C (10 mol %) and stirred under H₂atmosphere at room temperature for 2 h. after completion of thereaction, filtered through celite bed. The filtrate was removed underreduced pressure to afford the product.

Protein Preparations

Mouse SOX HMG fragments. The HMG domains of mouse SOX2 (Group B), SOX11(Group C), SOX6 (Group D), SOX9 (Group E), SOX18 (Group F) and SOX15(Group G) were BP cloned from cDNA templates (IMAGE cDNA clone IDs:Sox18: 3967084; Sox9: 5354229; Sox4: 6822618) into a pDONRTM221 pENTRYvector, sequenced and recombined into a pETG20A or a pHisMBP expressionplasmid using Gateway®LR Technology (Ng et al., 2012). Constructs weretransformed into Escherichia coli BL21(DE3) cells (Luria-Bertani, 100μg/ml Ampicillin).

Full-length mouse SOX18. A N-terminally tagged mouse HIS-GST-SOX18 wasrecombinantly expressed in Sf9 cells, purified on GST resin (GEHealthcare Life Sciences, Sweden) and eluted in Tris buffer (50 mM Tris,500 mM NaCl, 10 mM reduced glutathione, pH 8.0). cDNA clone of mouseSox18 was PCR amplified and cloned into the pOPIN-GST vector, togenerate N-terminally tagged HIS-GST-SOX18. A sequence verifiedconstruct was co-transfected with flashBACULTRA (Oxford ExpressionTechnologies, Oxford, United Kingdom) baculovirus DNA onto Spodopterafrugiperda Sf9 cells to obtain recombinantly expressed HIS-GST-SOX18.High Five cells (BTI-TN-5B1-4) in Sf-900™ II serum-free medium wereinfected at cell density of 1.5×106 cells/mL with a multiplicity ofinfection (MOI) of 5 PFU/cell, and incubated at 21° C. for 144 h beforeharvest. The cell pellet from 100 mL of expression culture wasresuspended in 30 mL of phosphate lysis buffer (50 mM sodium phosphate,500 mM sodium chloride, 1% Triton X-100, 2 mM magnesium chloride, onetablet of cOmplete Protease Inhibitor Cocktail, pH 7.5) and sonicated onice for 20 s. The cell lysate was centrifuged at 17,000×g for 40 min at4° C. Supernatant was incubated with Benzonase Nuclease (MerckMillipore) for 1 h at room temperature for DNA degradation, before beingmixed with 500 μL GST resin (GE Healthcare Life Sciences, Sweden) andincubated on a rotating wheel at room temperature for 1 h. The samplewas centrifuged at 500×g for 1 min to remove unbound protein in thesupernatant. The resin was further washed with 50 resin volumes (RV)wash buffer (50 mM sodium phosphate, 500 mM NaCl, pH 7.5), with unboundprotein removed by centrifugation as above. Bound protein was elutedfrom the resin with 3×1 RV elution buffer (50 mM Tris, 500 mM NaCl, 10mM reduced glutathione, pH 8.0), collecting the supernatant bycentrifugation as above.

DNA-Binding Competition Assay Using Fluorescence Polarization (FP)

The DNA-binding competition assay was performed in black 384-wellplates, with mouse full length SOX18, or SOX-HMG fragments. Allexperiments were performed using a fluorescently-labelled Prox1-DNAelement. Controls consisted of free labelled DNA (low FPmilli-Polarization index, mP), labelled DNA in presence of protein(negative control, high mP), and labelled DNA and protein in presence ofcompeting excess of unlabelled DNA (positive control, low mP)

The DNA-binding competition assay was performed in 25 uL, in black384-well plates, using either 30 mM HEPES(N-2-Hydroxyethylpiperazine-N′-2-Ethanesulfonic Acid) (pH 7.5, with 100mM KCl, 40 mM NaCl, 10 mM NH4OAc, mM Guanidinium, 2 mM MgCl2, 0.5 mMEDTA, 0.01% NP-40) for mouse full length SOX18, or Tris-NaCl (10 mM TrispH 8.0 and 100 mM NaCl) for SOX-HMG fragments. All experiments wereperformed using a 40 bp double-stranded Prox1-DNA element with 5′fluorescein amidite (FAM) label (Sigma Proligo or InVitrogen). Optimumbinding levels were obtained at 200 nM of mouse full length SOX18 and 60nM of SOX-HMG fragment, in presence of 5 nM labelled DNA. Controlsconsisted of free labelled DNA (low FP milli-Polarization index, mP),labelled DNA in presence of protein (negative control, high mP), andlabelled DNA and protein in presence of 400 time competing excess ofunlabelled DNA (positive control, low mP). Depending on compound, finalDMSO concentrations ranged from 2 to 3.33% v/v. After mixing protein,DNA probe and compound, plates were sealed and briskly agitated in thedark for 5 minutes at room temperature, 10 minutes at 37° C., and 30minutes at room temperature, before reading fluorescence polarization ona Tecan M1000Pro (λexc=485 nm, λem=525 nm). All experiments wereperformed in triplicates.

Cell-Based Functional Assay.

Monkey kidney fibroblast-like cells (COS-7) were cultured at 37° C., 5%CO2 in DMEM (Life technologies, 11995) with FBS, sodium pyruvate,L-glutamine, penicillin, streptomycin, non-essential amino acids andHEPES. Cells were grown in 96-well plates to 80% confluency, andtransfected with mouse plasmids pGL2 Vcam 1 promoter construct (VC1889)and pSG5 Sox18, using X-tremeGENE 9 DNA transfection reagent (Roche,06365787001) (Hosking et al., 2004, Duong et al., 2014). After 4-6 htransfection, cells were incubated with compounds in 0.5% FBS medium foranother 24 h, before lysis and luciferase assay (Perkin Elmer, 6016711).

Cell-Free Expression and ALPHAScreen.

Plasmid preparation and cell free-expression. All proteins weregenetically encoded with enhanced GFP (GFP), mCherry and cMyc (myc)tags, and cloned into cell free expression vectors (Gagoski et al.,2015, Sierecki et al., 2013). Translation competent Leishmaniatarentolae extract (LTE) was prepared as previously described toco-express protein pairs (Kovtun et al., 2011, Mureev et al., 2009).

Proteins were genetically encoded with enhanced GFP (GFP), mCherry andcMyc (myc) tags, and cloned into cell free expression Gatewaydestination vectors: N-terminal GFP tagged (pCellFree_G03), N-terminalCherry-cMyc (pCellFree_G07) and C-terminal Cherry-cMyc tagged(pCellFree_G08) (Gagoski et al., 2015). Human RBPJ (BC020780) and MEF2C(BC026341) Open Reading Frames (ORFs) were sourced from the HumanORFeome collection, version 1.1 and 5.1, and the Human Orfeomecollaboration OCAA collection (Open Biosystems), as previously described(Sierecki et al., 2013) and cloned at the ARVEC facility, UQ DiamantinaInstitute. The entry clones pDONOR223 or pENTR201 vectors were exchangedwith the ccdB gene in the expression plasmid by LR recombination (LifeTechnologies, Australia). The full-length human SOX18 gene wassynthesized and the transfer to vectors was realized using Gateway PCRcloning. Translation competent Leishmania tarentolae extract (LTE) wasprepared as previously described (Kovtun et al., 2011, Mureev et al.,2009). Protein pairs were co-expressed by adding 30 nM of GFP templateplasmid and 60 nM of Cherry template plasmid to LTE and incubating for 3hours at 27° C.

ALPHAScreen was performed as previously described (Sierecki et al.,2014, Sierecki et al., 2013). The assay for disruption ofprotein-protein interaction (IC50) was conducted by expressing theprotein pairs in LTE and incubating with a dilution range of testedcompounds (0.3 to 300 μM) or DMSO (0.7% DMSO final) in buffer B for 1 h.Percentage of interaction was calculated as: (I_cpd/I_DMSO)×100 from 3independent experiments.

ALPHAScreen was performed as previously described (Sierecki et al.,2014, Sierecki et al., 2013), using the cMyc detection kit andProxiplate-384 Plus plates (PerkinElmer). The LTE lysate co-expressingthe proteins of interest was diluted in buffer A (25 mM HEPES, 50 mMNaCl). For the assay, 12.5 μL (0.4 μg) of Anti-cMyc coated AcceptorBeads in buffer B (25 mM HEPES, 50 mM NaCl, 0.001% NP40, 0.001% casein)were aliquoted into each well. This was followed by the addition of 2 μLof diluted sample, at different concentration, and 2 μL of biotinlabeled GFP-Nanotrap in buffer A. The plates were incubated for 45 minat room temperature, then adding 2 μL (0.4 μg) of streptavidin-coatedDonor Beads diluted in buffer A, and incubation in the dark for 45 minat room temperature. The ALPHAScreen signal was measured on an EnvisionPlate Reader (PerkinElmer), using manufacturer's recommended settings(λexc=680/30 nm for 0.18 s, λem=570/100 nm after 37 ms). The resultingbell-shaped curve is an indication of a positive interaction, while aflat line reflects a lack of interaction between the proteins. Themeasurement of each protein pair was repeated in triplicate.

${❘{{The}{Binding}{Index}{was}{calculated}{as}:{BI}}} = {\left\lbrack \frac{I - I_{neg}}{I_{ref} - I_{neg}} \right\rbrack \times 100}$

I is the highest signal level (top of the hook effect curve) and Ineg isthe lowest (background) signal level. The signals were normalized to theIref signal obtained for the strongest interaction. The assay fordisruption of protein-protein interaction (IC50) was conducted byexpressing the protein pairs in LTE and incubating with a dilution rangeor single concentration of tested compounds (0.3 to 300 μM or 50 μM) orDMSO (0.7% DMSO final) in buffer B for 1 h. Percentage of interactionwas calculated as:

$\left( \frac{I_{cpd}}{I_{DMSO}} \right) \times 100.$

Data from 3 independent experiments were fitted in GraphPad Prismversion 6.0 using 3-parameter non-linear regression.

Co-Immunoprecipitation (Co-IP).

Co-immunoprecipitation was performed as described previously (Siereckiet al., 2014). Briefly, SOX18-Cherry-cMyc was co-expressed withGFP-RBPJ, GFP-MEF2C or a GFP construct as negative control bait, inLeishmania tarentolae cell-free protein expression system. NaCl wasadded to the expressed protein (200 mM) and the samples were incubatedwith 10 μL of GFP-nanotrap coated beads (NHS-activated sepharose coupledwith MBP-GFP-Nanotrap) for 30 min at 4° C. with gentle mixing byrotation. Subsequently, the beads were washed 6 times with 200 μL ofwash buffer (PBS with 0.1% Triton X-100 and 200 mM NaCl). The proteinswere released from the beads by heating for 3 min at 72° C. in 15 μL of2× NuPAGE LDS loading buffer and resolved on NuPage Novex 4-12% gel(Life Technologies, Australia). The gel was scanned for GFP and Cherryfluorescence using a ChemiDoc MP System (Bio-Rad, Australia).

Critical Micelle Concentration (CMC).

Critical micelle concentration was determined based on the incorporationof fluorescent 1,6-diphenyl-1,3,5-hexatriene (DPH) into micelles(Chattopadhyay and London, 1984). Small molecules and positive controls(neutral detergent Triton X100 and anionic detergent SDS) were cascadediluted in low binding 96-well plates from 1000 μM to 0.1 μM. Dilutionswere performed in 200 mM NaCl or FP buffer. DPH was supplemented at 5 μMinto black 384-well plates. Fluoresence intensity (λexc=360 nm, λem=430nm) was measured following a 30-min incubation at room temperature, tographically estimate CMC transition.

Cytotoxicity Assay.

Cell toxicity was determined using Alamar blue, as previously described(McMillian et al., 2002). COS-7 cells were seeded as 7000 cells per wellin black wall clear bottom 96-well plates in DMEM medium (LifeTechnologies Australia) with 10% FBS. HepG2 and HEK293 cell lines, fromATCC, were seeded as 5000 cells per well in black wall clear bottom384-well cell culture plates in DMEM with 1% FBS. Cells were culturedfor 24 h at 37° C., 5% CO2. A serial dilution of compounds was added,with a final DMSO concentration adjusted to 0.5% v/v. Cells wereincubated for another 24 h. 1 Triton X-100 was used as negative control,and 0.5% DMSO as positive control. 5 μM Alamar blue was added to eachwell and fluorescence was measured (λexc=560 nm, λem=590 nm) after 2 hincubation at 37° C. Data were analysed using Prism software.

Direct DNA-Binding Assay, Using Surface Plasmon Resonance (SPR).

Compounds were tested at a 1% v/v DMSO in HBS-EP buffer (10 mM HEPES,150 mM NaCl, 3 mM EDTA, 0.005% v/v polysorbate 20, pH 7.4). The samebuffer supplemented with 1% v/v DMSO was used as a mobile phase. DNAminor groove binder DAPI (4′,6-diamidino-2-phenylindole), DNAintercalator and minor groove binder actinomycin D, and DNA intercalatorethidium bromide were used as positive controls. Biotinylated (one tagper probe) double strand DNA probes were prepared using a standardannealing routine (5 min at 100° C., room temperature overnight andstored at −20° C.) of single strand anti-parallel DNA Probes purchasedfrom Geneworks and were immobilized on CM5-SA streptavidin chips as permanufacturer's recommendations. Running buffer flow was set at 25μL/min, cycles consisted of 4 min association, 4 min dissociation,followed with a pulse of 10 s of 10 mM Gly-HCl pH 2.5 for regeneration,and a 1 min stabilization, for compounds and actinomycin D. DAPI did notrequire any regeneration, whereas for ethidium bromide, a 40 s pulse at25 μL/min of 0.5% v/v SDS was required for regeneration beforestabilization. All sample and control cycles were performed intriplicate. DMSO calibration was performed as per manufacturer'srecommendations. After each injection, an extra flow system wash wasperformed with 50% v/v DMSO to avoid carryover. Experiments were run ona Biacore T200 (GE Healthcare, USA), with one flow cell kept as areference.

Direct Protein Binding Assay (Thermal Aggregation).

Differential static light scattering studies were conducted on aHarbinger Biotech StarGazer using mouse SOX18-HMG (109aa) in thepresence and absence of either Prox1-DNA or putative small moleculeligands. An initial experiment was conducted to evaluate the compoundconcentration for which the change in aggregation temperature was nolonger dependent on concentration. The final concentration that was usedranged from 500 μM (Sm4 and Sm14) to 1.5 mM (Sm5), depending on thenecessity to limit the concentration of DMSO to 3%. A 17 bp Prox1-DNAoligonucleotide was used at a final concentration of 10 μM. Binding wasperformed in triplicate and detected by an increase in Tagg (aggregationtemperature) of >2° C. in the presence of the ligand. Tagg was measuredwith the same protein batch in one single run. The reaction was carriedout in Tris-NaCl buffer (10 mM Tris-HCL, 150 mM NaCl, pH 8) with 3% v/vDMSO, in a final reaction volume of 45 μl and with a mouse SOX18-HMGconcentration of 154 μM. These were incubated for 1 h at roomtemperature before measurement. The protein was heated from 25° C. to °C. at a rate of 1° C. per min. Total intensities were plotted againsttemperature for each sample, and fitted to the Boltzmann equation bynon-linear regression.

In Silico Docking and Molecular Dynamics

Ligand-protein docking. In silico docking of Sm4 into the SOX18/DNAcomplex was performed using LeadIT/FlexX (BioSolveIT, Germany). Thedocking was performed by removing all the water molecules from thestructure of SOX18-HMG/Prox1-DNA (pdb: 4Y60) and defining the wholeprotein/complex as possible binding site. The best 20 docking poses ofSm4 were analysed and grouped into 4 clusters. As the SOX18/DNA does notcontain a classic binding pocket, each pose cluster was furthervalidated by a 200 ns long MD simulation. The MD simulation wasperformed using the full SOX18/DNA structure with Sm4 in its differentbinding pose. Analysis of the MD simulations revealed three of the posesas unstable, with the Sm4 breaking its interaction with the proteinwithin the first 3 to 14 ns of the simulation, remaining in the watersolvent for the remaining of the simulation. Only for one of the posesthe MD simulation produced a stable binding poses for Sm4 during theentire 200 ns simulation. Similarly, docking and MD simulations wereperformed with Sm4 and SOX18 without DNA. However, none of the 4-posecluster produced a stable binding orientation during the MD simulation.For comparison, MD simulations were performed for SOX18/DNA and SOX18without DNA, without Sm4, producing similar conformations and dynamicbehaviour as the structures with Sm4 ligand.

Protein-protein docking. The protein-protein docking between Notch1transcription complex and SOX18 was performed with ClusPro online serverversion (cluspro.bu.edu), using pdb: 3V79 and pdb: 4Y60 for thestructures of Notch1 transcription complex and SOX18-HMG, respectively.DNA molecules were removed before docking, as ClusPro is unable toprocess them, and restored after docking. Docking solutions withclashing DNA molecules were rejected. The resulting top docking pose wasused as starting conformation in a 50 ns long MD simulation to optimizethe docking pose, and validate the stability of the multi proteincomplex.

MD simulations. Any MD simulation was carried using the AMBER MDsoftware “pmemd”, using the ff99SB force field for the protein and DNA,TIP3 for implicit water, and applying periodic boundary conditions(NTP), particle-mesh Ewald (PME) method for long-range electrostaticinteractions, isotropic pressure coupling and Langevin thermostat(gamma_In=1/ps) for temperature coupling. The simulations were run with2fs step size, constraining bonds involving hydrogens using the SHAKEalgorithm. For MD simulations with Sm4, Antechamber from the AMBERpackage was used to calculate the force field parameter for the ligand.All MD simulations were performed by minimizing the structures andequilibrating them by reducing the position constrains slowly over 5 ns.Each simulation was done in triplicate, using different random numberfor the assignment of the initial velocities.

COX1/COX2 Enzymatic Assay

COX inhibition activity was measured using COX1 and COX2 inhibitorscreening assay kits from Cayman Chemical Company (Ref #701090 and701080). All compounds were tested in quadruplicate at a single 200 μMconcentration in 2% v/v final DMSO. Compounds were preincubated withovine COX1 or human COX2 at 37° C. for 10 min before incubation with COXsubstrate arachidonic acid for another 3 min. Reactions were stopped byaddition of concentrated hydrochloric acid. Prostanoid standard curvewas prepared and enzyme immunoassay performed as per manufacturer'sdescription. A DMSO control, a 100% inhibition control withheat-inactivated COX enzyme, as well as a 200 μM meclofenamate positivecontrol (potent non-selective COX1/2 inhibitor), were included forreference. Prostanoid standards values were plotted as logit (B/B0)versus log concentrations and fitted with linear regression usingGraphPad Prism version 6.0. Standard curve linear fit was used tocalculate each samples concentrations.

SOX18 Single Molecule Tracking

FIG. 24B depicts the experimental workflow, which involvesbi-dimensional tracking of molecule trajectory and analysis usingMATLAB, as outlined in Chen et al. (Cell 156, 1274-1285; 2014). We notethat the immobile DNA bound fraction (%, shown in pie graphs) is of twotypes, specific and non-specific, depending on the dwelling time in onesame DNA position; if of a short time (less than 1 second average) thisis considered to be non-specific binding (i.e., transient binding torandom DNA sites), and if of a long time (greater than 5-6 secondsaverage) this is considered to be specific binding (i.e., longer bindingto target DNA sites with transcriptional effect). The dwell times ofimmobile DNA bound fractions (shown as bar graphs) is also of two types,specific and non-specific: The length of time in seconds that SOX18molecules bind to DNA non-specifically (on average less than 1 second)or specifically (on average greater than 5-6 seconds). Single moleculetracking is performed after transfection of Hela cells with aSOX18-Halotag reporter construct. This expression vector enables us todetect single molecule of SOX18 protein upon addition of a ligand whichbecomes fluorescent after enzymatic processing by the Halotag system.Real time imaging is performed using a modified version of TIRFmicroscopy (HiLo) using a ZEISS ELYRA super-resolution microscope.

Results Screening of Natural Products for Inhibition of Sox18-DNABinding.

A marine extract library was screened for inhibitors of SOX18 protein(full-length murine) binding to DNA, using a fluorescencepolarization-based assay (FP). We selected a fluorescently labelledoligonucleotide harbouring a consensus SOX motif, found in the firstintron of Prox1 gene, a SOX18 direct target (Francois et al., 2008)(FIG. 1A). The library includes 2,000 purified metabolites, as well as2,688 marine extracts, containing in excess of 50,000 structures. Thisprimary screening identified sixteen active extracts collected fromvarious phyla, namely sponges (10), algae (5) and tunicate (1) (hit rate0.6%, primary screening Z′-factor=0.62) (Zhang et al., 1999). Subsequentextracts deconvolution was prioritized based on potency and abundance ofbiologically active molecule(s), with the most active extract, from thebrown algae Caulocystis cephalornithos, producing two active compounds:6-undecyl salicylic acid (Sm1), and 6-tridecyl salicylic acid (Sm2)(FIG. 1B). Dose response screening for both Sm1 and Sm2 resulted ininhibitory effects (IC50) in the high micromolar range (FIG. 1C). Bothactive compounds contain a salicylic acid scaffold with a largealiphatic group.

Design and Primary Screening of Focused Library.

In the next step, we designed a small library of analogues to validatethe salicylic acid (hydroxyl benzoic acid) active scaffold, andinvestigate its structure-activity relationship profile. The selection,shown in FIG. 2A, also included compounds with a similar resorcinolscaffold (replacing the carboxyl acid with an additional hydroxyl), aswell as a number of approved NSAID that contain a similar salicylic acidor anthranilic acid scaffold (replacing the hydroxyl with an amine). Thelibrary was purchased, and screened for inhibition of SOX18-DNA binding,using the FP assay. In this assay, disruption of high affinityprotein-DNA interaction requires an inhibitor concentration in the highmicromolar range, at which aggregation can occur, especially in the caseof amphiphilic and high logP molecules (Irwin et al., 2015). Therefore,the compounds were counter-screened for critical micelle concentration(CMC), to eliminate aggregate- or micelle-forming compounds as falsepositives (Table 1, columns 2 and 5, and FIG. 2B).

The CMC assay eliminated five compounds, Sm3, Sm6, Sm7, Sm9 and Sm10,displaying a CMC at 20 and 30 μM (Table 1, column 2). The remainderdisplayed no micelle formation up to 1000 μM and were included in theSOX18-DNA binding assay. The CMC of Sm1 and Sm2 could not be determineddue to short supply of compounds. The SOX18-DNA binding assay identified7 compounds with IC50 values below 1000 μM, with 2, Sm4 and Sm14,showing improved IC50 values of around 100 μM compared to the originalhits Sm1 and Sm2 (IC50 of around 350 μM) (Table 1, column 1; FIG. 2C).Interestingly, all three anthranilic acid analogues, meclofenamate,niflumic acid and flufenamic acid, also display activity with IC50values in the 100-400 μM range (Table 1, column 1; FIG. 2C).

In order to pinpoint the possible binding site of the small molecules,the FP assays were repeated with a shorter, DNA-binding centred proteinfragment. The DNA binding domain of SOX TFs consists of the 79 aminoacid long High Mobility Group (HMG) box. The 109 amino-acid long SOX18fragment (SOX18[109]) corresponds to residues 69-177 (numbering by mouseSOX18), which includes the HMG box (residues 78-149) and N- andC-terminal flanking sequences of 9 and 28 amino-acids, respectively. FPassay with the SOX18[109] fragment displayed almost identical IC50values to full length SOX18 (data not shown) suggesting that smallmolecules interfere with this 109 aa portion of the protein.

Binding Selectivity.

From a molecular viewpoint, an inhibitor of SOX18-DNA binding could acteither by interacting directly with the DNA, or the protein, or at theinterface between protein and DNA. Binding of small molecules directlyto the DNA, even though reported for other TFs (Leung et al., 2013), hasthe potential for unspecific DNA binding, leading to possiblegenotoxicity, mutagenicity, or carcinogenicity. For this reason, wedeveloped a direct binding assays to determine whether the activecompounds interact directly with DNA or protein. A surface plasmonresonance-based method was developed to analyse the binding of smallmolecules to biotinylated double stranded DNA immobilized on the surfaceof a streptavidin chip. The method measures the rate of binding (kon),dissociation (koff) and binding constant (KD), and was used to measurethe binding to two different DNA sequences: SOX binding site consensusDNA, and scrambled DNA. Intercalating agents ethidium bromide andActinomycin D, and the minor groove-binding agent4′,6-diamino-2-phenylindole (DAPI) were used as positive controls forunselective DNA binding. All positive controls displayed KD independentof DNA sequence and consistent with literature. This analysis showedthat none of the SOX18 inhibitors displayed any binding to eitherconsensus or scrambled DNA (FIGS. 3A and 3B).

To investigate whether the inhibitors interact directly with theprotein, we measured the resulting increase in SOX18 thermostability, byassessing the shift to higher temperature of protein unfoldingequilibrium (Shrake and Ross, 1992, Shrake and Ross, 1990, Brandts andLin, 1990, Fukada et al., 1983). For this, we used static lightscattering as a readout of protein-inhibitor complexes aggregation(Mittal et al., 2014, Senisterra et al., 2006, Senisterra et al., 2008,Senisterra and Finerty, 2009, Senisterra et al., 2012). Thermalstability was measured using HMG only SOX18[109] fragment, and a DNAprobe decorated with SOX motifs, as positive control. Sm4, Sm5 and Sm14were tested at concentrations saturating all SOX18 potential bindingsites, respectively 500 μM for Sm4 and Sm14 and 1.5 mM for Sm5. In thesemaximum ligand binding conditions, temperature-dependent proteinaggregation is no longer limited by binding site occupancy and wasmeasured until a plateau was reached. Each inhibitor displayed anincrease in Tagg of more than 3° C., consistent with direct interactionof inhibitors to protein (FIG. 3C). Taking both binding studiestogether, results suggest that small molecule inhibitors interact withSOX18 protein, without interacting directly with DNA. In addition,combined data from the FP assay and the thermal stability assay suggestthat the inhibitor-protein interaction site is located in or in closeproximity of the SOX18-HMG box.

Sox DNA-Binding Inhibition Selectivity.

The DNA binding domains of all SOX proteins are highly conserved,sharing 46% sequence identity with the HMG domain of mammaliantestis-determining factor SRY (Bowles et al., 2000, Gubbay et al.,1990), while the remaining domains of the SOX TFs, flanking the HMGdomain, show low levels of similarity. To investigate the selectivity ofthe SOX18 inhibitors, HMG only protein fragments from different SOXproteins were used in the DNA-binding FP based assay, using afluorescently labelled oligonucleotide harbouring a known SOX motif,5′AACAAT3′. The different SOX TFs include: SOX2 (Group B), SOX11 (GroupC), SOX6 (Group D), SOX9 (Group E), SOX18 (Group F), and SOX15 (GroupG). The most active inhibitor Sm4 was assessed against all different SOXTFs, displaying DNA-binding inhibition in all cases (IC50 around 200-300μM) with a slight preference for SOX18 and SOX15 (IC50 around 200 to 220μM versus 270 to 310 μM for others), suggesting that for DNA-bindingdisruption the inhibitor is non selective amongst SOX TFs (FIG. 3D).

Off Target Analysis.

COX inhibition. Salicylates are an important class of NSAIDs acting viadirect or indirect suppression of cyclooxygenase (COX) dependentproduction of pro-inflammatory prostaglandins (Pillinger et al., 1998).In this study, we identified SOX18 inhibitors with structuralsimilarities to COX inhibitors, and in order to investigate anyfunctional overlap between NSAID and the novel SOX18 inhibitors, weincluded structurally similar NSAID in the SOX18 investigation.Similarly, we investigated whether novel SOX18 inhibitors would inhibitCOX1/2 enzymatic activities. COX-1 and COX-2 inhibitory effects of Sm4,Sm5, Sm8, Sm11, Sm12, Sm13 and Sm14 were assessed using a commercialCOX-1/2 ELISA assay, and using meclofenamic acid as positive control.None of the SOX18 inhibitor display any COX-1 or COX-2 inhibitoryactivity up to a concentration of 200 μM.

Off-target profiling. We further explored potential SOX18-independenteffects of our lead Sm4 with a Eurofins-CEREP/Panlabs panel ofradioligand binding assays to various receptors, enzymes andtransporters, including G-protein-coupled receptors (GPCRs), ionchannels, membrane receptors, kinases and non-kinase enzymes, andnuclear receptors, involved in a broad range of potential off-targeteffects (Table 4). A number of epigenetic modifiers were also testedusing a series of recombinant-enzyme fluorimetry assays. No significantinhibition (>50%) was observed at 10 μM, demonstrating Sm4 selectivityand potential for further drug development.

Sox18 Protein-Protein Interaction

The highly conserved HMG domain of SOX transcription factors has beenreported to be involved in both protein-DNA and protein-proteininteraction (Agresti and Bianchi, 2003, Prokop et al., 2012, Huang etal., 2015). Hence, compounds that bind directly to or in close proximityof the HMG domain have the potential to modulate both SOX18-DNA andSOX18-protein interactions, either directly or by allosteric changes tothe conformation of the SOX18 protein. To investigate the potential ofour small molecules to modulate SOX18-protein interactions, we selectedtwo SOX18 PPIs known to be involved in the transcriptional regulation ofendothelial cells: MEF2C, reported to bind to SOX18 in a GST-pull downassay (Hosking et al., 2001), and RBPJ, reported to interact geneticallywith SOX18, while no direct binding has been identified yet (Sacilottoet al., 2013). In addition, XRCC6 (ATP-Dependent DNA Helicase II) wasselected as negative control, as a non-binding partner.

We analysed PPI inhibition using a cell-free expression system toexpress tagged proteins, and combined it with ALPHAScreen technology(Amplified Luminescent Proximity Homogeneous Assay) enabling us tomeasure tagged proteins propinquity (Sierecki et al., 2013). Thisapproach confirmed direct pairwise interaction between SOX18 and itsknown partner MEF2C, and revealed a direct PPI between SOX18 and RBPJ(FIG. 4A, Left Panel). Direct physical interaction was further validatedusing standard co-immunoprecipitation (FIG. 4A, Right Panel). Next, weinvestigated the ability of our most potent small compound, Sm4, as wellas meclofenamic acid, niflumic acid and flufenamic acid, to disruptSOX18-MEF2C or SOX18-RBPJ interactions. Sm4 disrupts SOX18-RBPJinteraction with an IC50 of 42.3 μM, but has no effect on SOX18-MEF2Cinteraction (FIG. 4B, Top Left and Top Right Panels). Conversely,flufenamic disrupts the SOX18-MEF2C interaction (IC50 of 29.1 μM), whileonly weakly the SOX18-RBPJ interaction (IC50 of ˜444 μM) (FIG. 4B, TopLeft and Bottom Right Panels). The other NSAID showed little or noeffect on any of the PPI.

SAR Library.

The structure-activity relationship (SAR) of the salicylic acid typeinhibitors of SOX18-DNA binding and SOX18/RBPJ PPI has been investigatedin more detail with a separate library of analogues. This library wasdesigned to query the significance of some of Sm4 and other salicylatesdistinctive features, including: electron density of the salicylatearomatic ring, significance of the two acidic hydrogen of the 11hydroxyl carboxylic acid motif, saturated- or ethylene-linkage withlipophilic tail, as well as structure and lipophilicity of thelipophilic tail. We synthesized 30 analogues, Sm15-Sm44 (FIG. 5 ), whichwere screened for inhibition of DNA binding to the HMG only SOX18[109]fragment and disruption of SOX18-RBPJ interaction (upper and lowerpanels). Additionally, a number of these analogues (i.e., Sm4, Sm17 toSm24, Sm26, Sm31, Sm34, Sm37 and Sm40 to Sm42) were also tested fordisruption of SOX18-SOX18 homodimerization interactions using theaforementioned ALPHAScreen assay. As shown in FIG. 5 , Sm4, Sm17 toSm23, Sm26, Sm31, Sm34, Sm 37, Sm40 and Sm41 demonstrated someinhibitory activity in respect of SOX18-SOX18 homodimerization at aconcentration of 5 uM. This library was also screened for generalcytotoxicity against two cell lines, HEK293 and HepG2, to evaluate theirdevelopment potential (Table 3).

Potential PAINS (Pan Assay Interference Compounds) chemical moieties,common in promiscuous frequent hitters, which act as false positives inmany biochemical high throughput screens, were analysed using the insilico predictor “FAF-Drugs3”(http://fafdrugs3.mti.univ-paris-diderot.fr/) (Baell and Holloway, 2010,Lagorce et al., 2008). In total only 3 compounds were flagged with PAINSmoiety (Sm14, Sm40 and Sm44). Sm14 contains a methylene-thiazolonemotif, or reactive α,β-unsaturated carbonyl group, while both Sm40 andSm44 contain a oxidative labile catechol group (Table 2). While Sm40 andSm44 were not active in the FP assay, Sm14 was not pursued beyond thebinding assays. In addition, the new library was analysed for potentialaggregators using the “Aggregator Advisor” database, however none showedsimilarity to known aggregator and all showed moderate lipophilicity,with logP values below 5.8 (Table 3).

SAR for SOX18-DNA binding inhibition. Activity data display some clearSAR for SOX18-DNA binding inhibition, with a reduction or elimination ofactivity by any etherification, esterification or amidification of bothor either one of 11 hydroxyl or carboxylic acid. Interestingly,replacement of the carboxylic acid with hydroxyl is tolerated, eventhough it reduces the inhibitory activity of the compounds. Similarly,salicylic acids para-substituted with small electron donating groups aretolerated displaying similar activity; however, the reduced acidityseems to increase cytotoxicity. For the aromatic tail, replacing thenaphtyl with a phenyl (Sm22) completely eliminated all activity. Fromthis small library, only Sm20, an unsaturated analogue of Sm4, displayedSOX18-DNA binding inhibiting activity and low cytotoxicity, similar toSm4 (FIG. 5 ).

SAR for SOX18-RBPJ protein-protein binding inhibition. Analogues werefirst tested at 50 μM. Compounds displaying more than 50% inhibitionwere then retested at a lower 5 μM. Activities are compared to controllevels of SOX18-RBPJ in presence of vehicle solvent with or without leadcompound Sm4, also tested at both concentrations. As expected from IC50plot depicted in FIG. 4B (Top Right Panel), inhibition by Sm4 at 50 μMis almost complete (11.9±5.6%, FIG. 5 , Bottom Panel) and marginal at 5μM. At high concentration of 50 μM, half of all tested compoundsdisplayed strong activity, however, only Sm18, Sm19, Sm26, Sm34 and Sm40remained highly active at 5 μM, with Sm26 remaining moderately active.Interestingly, none of these five potent PPi inhibitors, inhibitprotein-DNA binding as well.

The pattern in the inhibition of SOX18-RBPJ protein-protein interactionis less clear as in the inhibition of SOX18-DNA interaction. Several ofthe compounds containing the vinyl naphthalene are active in PPIinhibition; it is however, unclear whether the activity is due toincrease rigidity of the linker or due to the reactivity of thevinyl-aromatic group as Michael acceptor. A distinct feature of thislibrary is that the free carboxylate is not required for PPI activity,as di-ethyl amides or poly hydroxyl/methoxy containing compounds showPPI inhibition at 50 μM, however no inhibition of the DNA binding.Lastly, there is little overlap between PPI and DNA-binding inhibition,with the exception of Sm4 and Sm20, which inhibit both DNA and PPI.

Structural Investigation

The three-dimensional structure of mouse SOX18-HMG domain bound to DNAharbouring a SOX motif has been recently resolved by X-raycrystallography (Klaus et al., 2016), showing high similarity to otherHMG domains. However, attempts to co-crystallize SOX18 HMG domain boundto DNA in presence of Sm4 failed to identify a binding pocket for theinhibitor, as no electron density for the inhibitor could be detected.This was likely due to the protein/DNA disruption properties of Sm4. Toevaluate possible binding sites for Sm4, we used in silico docking andmolecular dynamics calculation, using the SOX18/DNA crystal structure.In the absence of a defined binding pocket in the SOX18-HMG domain, thein silico docking produced several possible binding poses, which werefurther validated by molecular dynamics (MD) simulations. Thesesimulations identified one binding pose that remained stable during theentire simulation time of 200 ns. In comparison, in all other poses theinhibitor broke its protein interaction after 3 to 14 ns, remaining inthe surrounding solvent without protein contact. Similarly, no stableSm4 binding poses were found using the SOX18-HMG structure without DNA.

The stable binding pose for Sm4 in the SOX18/DNA structure puts theinhibitor in a solvent accessible pocket between protein and DNA that isotherwise occupied by water molecules in the X-ray crystal structure.Sm4 main polar interactions are with Arg136 and Lys147, which swaps itsinteraction with dG15, and with some induced conformational changes toHis143, rotating its side chain towards Sm4 (FIG. 6A, B). However, noother major conformational changes could be observed that would directlyexplain Sm4's mode of DNA binding inhibition.

To investigate possible protein-protein interaction sites of SOX18, weused in silico protein-protein docking, in combination with MDsimulations, to build a complex model of SOX18/DNA with its proteinpartner RBPJ. For RBPJ we used the X-ray crystal structure of a sectionof the human Notch transcription complex, elucidated in 2012 (Choi etal., 2012). This section contains the ankyrin (ANK) repeat domain, theRBPJ-J-associated molecule (RAM) domain of the Notch intracellulardomain, bound to coactivator MAML1, as well as the transcription factorRBPJ bound to its consensus DNA. Docking the SOX18/DNA structure intothe structure of this Notch transcription complex with subsequent MDsimulation for optimization, resulted in a complex model shown in FIG.6C. This model indicates that RBPJ/SOX18 complex can be mediated by theHMG domain and is able to form a protein complex with no interference byboth DNA molecules. Indeed, both DNA strands, from RBPJ and SOX18, areorientated nearly parallel to each other, similar to the DNA orientationon a nucleosome. In addition, both C and N-terminal tails of the HMGdomain are orientated towards the solvent, allowing addition of themissing SOX18 domains without immediate interference with the RBPJcomplex.

The interaction between SOX18 and RBPJ is provided by the C-terminalpart of helix 3 and residues from the C-terminal tail (residues Gln138,Arg141, Asp142 and His143) of the HMG domain. This protein-proteininterface is thereby opposite to the DNA-binding interface of thatregion. Mapping the putative binding site of Sm4 onto the RBPJ/SOX18complex positions the inhibitor right into SOX18 DNA-binding region ofhelix-3 and C-terminal tail, opposite its main protein-proteininterface, suggesting the possibility that binding of Sm4 could perturbboth protein-protein and protein-DNA interactions.

Modulation of Sox18 Transcriptional Activity

To further assess the functional effects of the SOX18 inhibitors, weused an in vitro cell-based reporter system, as a readout of SOX18transcriptional activity. COS-7 cells were transfected with constructscontaining a Vcam-1 promoter fragment fused to a luciferase reportergene and a SOX18 expression vector. Sm4, our lead compound in term ofPPI disruption specificity was tested in this cell-based assay, alongwith meclofenamic, niflumic and flufenamic acids. Of these four smallmolecules, Sm4 displayed the most effective inhibition of SOX18transcriptional activity, with an IC50 value of 5.2 μM (FIG. 6D).Meclofenamic acid and flufenamic acid cytotoxicity was reached beforeany concentration-dependent SOX18 inhibition could be observed. Allother tested compounds displayed lower potency (20 μM<IC50<50 μM, Table1, column 3).

The observation that Sm4 selectively perturbs a particular PPI in aconcentration range similar to the one required to inhibit itstranscriptional activity in vitro suggests that the mode of action ofthis small compound is likely to be via interference with proteinpartner recruitment.

Assessing SOX18 target engagement by SM4 using Single Molecule Tracking(SMT). SMT technology enables us to visualize in real time in live cellnuclei the search pattern of the SOX18 protein for its target genes onchromatin at a single molecule of resolution. The fact that it ispossible to visualize SM4 perturbation on SOX18 chromatin bindingdynamics is a clear demonstration of SM4 on-target engagement. Thiseffect is observed at concentrations devoid of any cytotoxicity. Theeffect of SM4 causes SOX18 to dwell longer on the chromatin at specificsites, this change in the protein dynamics is likely to be theconsequence of changes in SOX18 protein-protein interaction which isimpaired by SM4 (as shown previously by the ALPHAScreen assay).Transcription factor mode of action is driven by a code ofprotein-protein interaction that instructs gene target selectivity. Ifthis code is altered the transcription factor activity is invalidated.

Discussion

Transcription factors are proteins that have a DNA-binding domain,multiple protein partners, and in some cases even an endogenous ligand.The notion that TFs rely on this type of interactions for their activityopens up different avenues for the discovery of molecules modulatingtheir function, such as screening for protein-DNA or protein-proteininteraction inhibitors. While there is a wealth of information on thegenetic pathways in which TF are involved, little is reported on theirmolecular mode of action, and more particularly, on the recruitment ofmultiple protein partners. Hence, screening for DNA binding inhibitorshas, until recently, provided the main option to find TF modulators,even though DNA-binding domains are highly conserved within TF familiesand constitute a region with low potential for selectivity.

In this study, we used a high-throughput DNA-binding assay to screen achemically diverse natural products library, identifying compounds ableto inhibit the DNA-binding of transcription factor SOX18. The screeningidentified two compounds of similar structure, both with a salicylicacid core and a lipophilic tail. A focused library designed around thetwo active compounds identified a wider range of similar compounds, withvarying degrees of activity, proving that the compounds form a clusterof SOX18 DNA-binding inhibitors. With the focused library, we furtherdemonstrated that the inhibitory activity was due to binding of thesmall compounds to the protein and not to the DNA itself. The activemolecules interact with the SOX18 protein, increasing its thermalstability upon binding. In addition, using either SOX18 full length orHMG box-only proteins, we proved that the compounds interact directlywith the DNA-binding domain or in its immediate vicinity.

We argued previously that using disruption of DNA-binding as a filterwould yield compounds with low selectivity between SOX transcriptionfactors due to a high level of sequence conservation of the HMG-box. Inagreement with this, our lead compound, Sm4, was able to disrupt the DNAbinding activity of a wide range of HMG-box from various SOX proteinswhen used at high concentration (200-300 μM). The HMG box of SOXproteins consists of three α-helixes, with two providing the maininterface for DNA-binding. However, SOX9 HMG box has been reported to beinvolved in protein-protein interaction as well (Huang et al., 2015,Agresti and Bianchi, 2003, Prokop et al., 2012), with the third α-helixproposed as the main interface for the partner proteins. In this study,we applied an in vitro method of protein-protein interaction (PPI)detection to investigate the disruption of TF protein partnerrecruitment. Direct protein-protein interaction for transcription factorSOX18 has only been reported for MEF2C (Hosking et al., 2001), while agenetic interaction with RBPJ has only been shown in the transactivationof D114 gene (Sacilotto et al., 2013). Both proteins display directbinding to SOX18 in the Cell-free/ALPHAScreen and Co-IP assay.Importantly, some of the small molecules differentially disrupt specificPPIs. Sm4 with a salicylic acid scaffold is, thereby, more selective indisrupting the SOX18/RBPJ complex, whereas flufenamic acid with ananthranilic acid scaffold is more selective for the SOX18/MEF2C complex.Further, in silico docking provides a putative binding pocket for Sm4,close to the C-terminal tail of the HMG box, wedged in between DNA andthe third helix of the protein. This location suggests that an inhibitorlike Sm4 would be able to alter the conformation of the SOX18 protein tonot only affect DNA binding, but also the interaction surfaces withprotein partner such as RBPJ.

Further investigation of the effect of small compounds on SOX18transcriptional activity, revealed that Sm4 is able to blockSOX18-dependent Vcam-1 promoter activity, when fused to a luciferasereporter gene. Flufenamic acid displays only little effect on SOX18transcriptional blockade. This reporter assay was conducted in COS-7cells that were transfected with both Sox18 and luciferase expressionvectors, hence limiting the interpretation of a potential disruption ofSOX18 endogenous partner recruitment. Nevertheless, the inhibition ofSOX18 regulated transcription by Sm4 is a clear indicator that a smallmolecule can interfere with a transcription factor activity in acell-based environment.

Synthesis and screening of an extended library further indicated someclear structure-activity relationships for inhibiting the SOX18/DNAbinding. While some degree of variation is tolerated in the lipophilictail and its linker, both hydroxyl or carboxylic acid groups have toretain their hydrogen-bond donating capabilities, as any esters, ethersor amides abolish the activity. Variation of the carboxylic acids withpara substituted electron donating groups has little effect, as does thereplacement of the acid with hydroxyl (resorcinol scaffold), bothretaining their ability to inhibit SOX18 DNA binding. Compounds with ananthranilic acid scaffold were selected as an extension of the chemicalsimilarity of the salicylic acid scaffold to NSAID compounds. While thestudy showed that, SOX18 inhibitors have no inhibitory activity againstCOX1 or COX2, some NSAID compounds display SOX18 DNA binding inhibition.However, difference in SOX18-protein binding inhibition (SOX18-MEF2Cinhibition instead of SOX18-RBPJ) suggests a different mode of bindingor action.

The efficacy of a compound to inhibit the transcriptional activity of aTF depends on the concentration of both TF and compound in the nucleus.The concentration of TFs can reach almost millimolar levels in thenucleus (Chen et al., 2014), while compound concentration depends on itsability to partition through both cell and nucleus membranes. Forinitial drug discovery it is more informative (i.e. to build SAR models)to measure the IC50 in homogenous assay, where the compoundconcentration is defined. However, for further drug optimisation thepenetration of the compound into the nucleus needs to be considered aswell, either with predictive models or with cell-based assays, measuringthe effective inhibition concentration (IC50) of compounds.

The other consideration to be made when developing TF inhibitors iswhich PPIs are predominately affected by the compound. Protein-proteininteractions, including interaction between TFs, are relatively weak.For example, the interaction between p32 and HDM2 has a KD in the low tomid micromolar range (Dawson et al., 2003, Chen et al., 2013). Incomparison, antibody-antigen interactions or interactions betweenendogenous peptide ligand and receptors (e.g. EGF-EGFR) are muchstronger, with KD in the low nanomolar to high picomolar range (Mian etal., 1991, Lax et al., 1988). Similarly, interactions between proteinand DNA are in the low nanomolar range, mostly due to strongelectrostatic interactions between negatively charged DNA and a usuallypositively charged DNA-binding domain. Sm4 is able to inhibit bothSOX18/DNA and SOX18/RBPJ interactions, however, the inhibitory effect isgreater on the weaker PPI.

An important consideration in the development of TF inhibitors is theability to selectively inhibit PPI, especially since TFs are capable ofrecruiting half a dozen different protein partners (Gamper and Roeder,2008). Even though TFs are intrinsically disordered (Wright and Dyson,2015), these proteins display domains modularity for differentprotein-protein interfaces (Reichmann et al., 2005). This implies thatsome interactions share structural similarities, while activatingdifferent downstream pathways. Similar to other regulatory proteins andenzymes, such as kinases, the selectivity profile of TF protein-proteininhibition needs to be considered. While blockade of all PPIs might notbe desirable, maximum efficacy might not be achieved with single PPIinhibition, but by simultaneously inhibiting the recruitment of a subsetof selected protein partners.

In conclusion this study identified salicylic acid derivatives as amajor pharmacophore for SOX18 inhibition, and Sm4 as a lead compound.Future drug-optimization should be performed following cues from bothprotein-DNA binding and PPI assays to further refine selectivity andpotency.

TABLE 1 Focused library compounds ability to inhibit SOX18-DNA bindingand SOX18-dependent transactivation in vitro. In vitro efficacy in Invitro cell cytotoxicity FP IC₅₀ ± SD CC₅₀ ± SD Aggregator IC₅₀ ± SD CMCμM μM advisor Compound μM μM COS7 (Luc) COS7 logP Natural products Sm1  348 ± 1.1  — — — 6.8^(#) Sm2   341 ± 1.1  — — — 7.8^(#) Salicyclic acidanalogues Sm3  — 30 — — 6.1^(#) Sm4  97.5 ± 1    >1000 5.2 ± 1.1  117 ±29.4 4.6^(#) Sm5  1,105 ± 1     >1000 21 ± 14 >200 3.2^(#) Sm6  — 20 — —6.1^(#) Sm7  — 20 — — 6.3^(#) Sm8   327 ± 1.1  >1000 ~50 >200 5.5^(#)Sm9  — 20 — — 4.8^(#) Sm10 — 20 — — 6.1^(#) Resorcinol analogues Sm113,803 ± 1    >1000 ~50 >200 2.6* Sm12  2,880 ± 1.1   >1000  33 ± 3.798.6 ± 17.4 3.8* Sm13 481 ± 1.1 >1000 ~30 >200 3.2^(#) Sm14 120 ±1.1 >1000 25 ± 9  >200 No aggregation NSAID analogues Salicylic — — — —No acid aggregation Aspirin — — — — No aggregation Gentisic — — — — Noacid aggregation Meclofenamic  163 ± 17.7 — >>CC₁₀  16 ± 0.3 5.6^(#)acid Nifumic acid 375 ± 84  — 50.5 ± 7.6  >200 3.4^(#) Flufenamic  220 ±68.6 — >CC₁₀  65 ± 2.7 4.8^(#) acidWith respect to Table 1 above, in the first column, compounds FP IC50were estimated using variable Hill slope curve fitting. Differentconcentration ranges were tested in the FP-based DNA binding competitionassay (0.2-200 uM, 10-500 uM, 10 uM-3 mM), with DMSO concentrationsranging from 0 to 3.33% v/v. Experiments were performed in threeindependent replicates. The second column summarizes thresholdconcentrations at which compounds Sm1-14 start forming micelles insaline (200 mM NaCl) or fluorescence polarization buffer (30 mM HEPES pH7.5, 100 mM KCl, 40 mM NaCl, 10 mM NH4OAc, 10 mM Guanidinium, 2 mMMgCl2, 0.5 mM EDTA, 0.01% NP-40). The third and fourth columns summarise50% inhibitory concentration -IC50- of cell-based luciferaseSOX18-dependent transactivation as well as cytotoxicity -CC50- for allactive compounds, in COS7 fibroblasts. In the last column, we used“Aggregator Advisor” (Irwin et al., 2015) to predict aggregators, basedon physical properties (CLog P>3) and likeness to a 12,600compound-strong library of known aggregators (*: compound similar toknown aggregator, #: not similar to any known aggregator, but possiblerisk because of “high” LogP.: no predicted risk of aggregation).

TABLE 2 PAINS analysis Evaluation of compounds Sm1-Sm44 for containingPAINS substructures, listing the three compounds found with possiblepromiscuous binding structure. Compound PAINS Substructure Comments

Methylene-thiazolone motif: a frequent hitter in biophysical assays,that can be further oxidised into a reactive metabolite, and ispotentially a CYP450 covalent binder.

Catechol motif: associated with a risk of further oxidation intoortho-quinones, potential covalent binder, especially to CYP450.

TABLE 3 DNA-binding and cytotoxicity Experimental data for Sm15-Sm44 forinhibition of SOX18/DNA binding (FP) and cytotoxicity against twomammalian cell lines. Also shows the predicted lipophilicity as cLogP.FP bound fraction (%) Cytotoxicity (CC₅₀ μM) Cmpd 200 μM 50 μM HEK 193HepG2 cLogP Sm15 120.8 ± 4.7   102.6 ± 7.4   >100 59.1 5.2 Sm16 108.9 ±4.9   99.3 ± 6.2  >100 45.1 5.2 Sm17 117.0 ± 10.9  100.3 ± 6.0   62.638.5 4.8 Sm18 106.9 ± 21.3  98.7 ± 5.0  38.7 28.0 4.7 Sm19 107.6 ± 4.5  99.2 ± 6.8  >100 >100 5.0 Sm20 11.9 ± 8.5  10.3 ± 4.1  97.1 71.3 5.0Sm21 98.1 ± 2.1  103.4 ± 8.8   >100 >100 3.8 Sm22 93.1 ± 9.5  98.1 ±6.1  >100 >100 3.4 Sm23 106.4 ± 1.7   102.5 ± 9.7   36.0 20.3 5.2 Sm24102.8 ± 16.6  97.4 ± 9.0  30.6 36.5 5.2 Sm25 106.8 ± 0.4   97.3 ± 8.2 85.8 35.8 4.8 Sm26 128.3 ± 17.2  98.5 ± 5.9  23.5 16.8 4.2 Sm27 92.8 ±21.7 97.0 ± 6.1  >100 >100 5.0 Sm28 19.3 ± 8.4  76.3 ± 2.3  65.1 78.95.0 Sm29 2.0 ± 7.2 73.0 ± 1.9  64.4 71.2 4.6 Sm30 109.4 ± 2.8   99.0 ±7.6  >100 >100 4.6 Sm31 91.7 ± 2.1  97.7 ± 5.9  92.7 >100 5.7 Sm32 10.9± 5.2  3.9 ± 6.0 37.6 24.0 5.6 Sm33 9.9 ± 2.3 50.1 ± 54.6 33.4 34.5 5.2Sm34 100.9 ± 1.5   94.2 ± 1.9  >100 >100 5.4 Sm35 −3.7 ± 7.9   −1.5 ±3.5   52.2 30.3 5.4 Sm36 −3.9 ± 6.8   10.9 ± 9.6  39.9 37.7 5.0 Sm37103.5 ± 10.4  98.2 ± 7.4  >100 >100 4.1 Sm38 119.5 ± 6.6   101.6 ±11.2  >100 >100 4.1 Sm39 116.6 ± 8.1   101.7 ± 9.7   >100 >100 3.6 Sm4092.7 ± 14.7 101.0 ± 12.1  >100 >100 2.7 Sm41 90.2 ± 46.1 100.3 ±10.0  >100 >100 5.3 Sm42 104.5 ± 7.7   100.7 ± 7.2   >100 >100 5.5 Sm43113.1 ± 3.6   105.5 ± 9.5   >100 >100 4.7 Sm44 53.5 ± 9.6  107.6 ±10.1  >100 >100 3.8

TABLE 4 Off-target activity profile of Sm4 Off-target activity profilefor Sm4 at 10 uM, using Hit Profilingscreen ® package from EurofinsCEREP/Panlabs (France, USA, Taiwan) Inhibition at 10 Assay Name SpeciesFamily Sub-Family μM (%) Adenosine A1 Human GPCR Adenosine −8 AdenosineA_(2A) Human GPCR Adenosine 30 Adrenergic α_(1A) Rat GPCR Adrenergic 0Receptors Adrenergic α_(1B) Rat GPCR Adrenergic −1 Receptors Adrenergicα_(2A) Human GPCR Adrenergic 0 Receptors Adrenergic β₁ Human GPCRAdrenergic 6 Receptors Adrenergic β₂ Human GPCR Adrenergic 2 ReceptorsCannabinoid Human GPCR Cannabinoid 3 CB₁ Dopamine D₁ Human GPCR Dopamine12 Dopamine D_(2s) Human GPCR Dopamine 15 Histamine H₁ Human GPCRHistamine 2 Muscarinic M₂ Human GPCR Muscarinic 13 Muscarinic M₃ HumanGPCR Muscarinic 8 Opiate μ (OP3, Human GPCR Opioid & Opioid- 3 MOP) likeProstanoid EP₄ Human GPCR Prostanoid 27 Serotonic 5- Human GPCRSerotonin −2 HT_(2B) Calcium Rat Ion Channels Ca2+ Channels −8 ChannelL-Type GABA_(A), Rat Ion Channels GABA Channels −6 FlunitrazepamGABA_(A), Rat Ion Channels GABA Channels 6 Muscimol Glutamate, Rat IonChannels Glutamate Channels −5 NMDA Nicotinic Human Ion ChannelsNicotinic Channels 3 Acetylcholine Potassium Hamster Ion Channels K+Channels 10 Channel [K_(ATP)] Potassium Human Ion Channels K+ Channels−14 Channel hERG Sodium Rat Ion Channels Na+ Channels −13 Channel, Site2 Phorbol Ester Mouse Kinases AGC 15 Nicotinic Human Nicotinic NicotinicChannels −4 Acetylcholine Channels α1 Imidazoline I₂ Rat Non-KinaseImidazoline 11 enzymes Rolipram Rat Non-Kinase Phosphodiesterases 1enzymes Androgen Human Nuclear Steroid NR 1 (Testosterone) ReceptorsEstrogen Human Nuclear Steroid NR −10 ERalpha Receptors GlucocorticoidHuman Nuclear Steroid NR 12 Receptors Thyroid Rat Nuclear Non-steroid NR36 Hormone Receptors Sigma1 Human Other Sigma −6 ReceptorsNorepinephrine Human Transporters Norepinephrine 34 (NET) HDAC3 HumanEpigenetics HDACS −4.8 HDAC4 Human Epigenetics HDACS 5.1 HDAC6 HumanEpigenetics HDACS 1.1 HDAC11 Human Epigenetics HDACS −29.1 Sirtuin 1Human Epigenetics HDACS 0.0 Sirtuin 2 Human Epigenetics HDACS −5.2

TABLE 5 Chemical characterization of synthesized compounds Sm15- Sm44High Res. HPLC Molecular Chemical MS Calc. Found Purity by Cmpd FormulaName Mass Mass UV 254 nm NMR Sm15 C₂₄H₂₅NO₂(Z)-N,N-diethyl-2-methoxy-6-(2- 359.189 360.234 >95 ¹H(naphthalen-2-yl)vinyl)benzamide [M + H] ¹³C Sm16 C₂₄H₂₅NO₂(E)-N,N-diethyl-2-methoxy-6-(2- 359.189 360.234 >95 ¹H(naphthalene-2-yl)vinyl)benzamide [M + H] Sm17 C₂₄H₂₇NO₂N,N-diethyl-2-methoxy-6-(2- 361.204 362.250 >95 ¹H(naphthalene-2-yl)vinyl)benzamide [M + H ¹³C Sm18 C₂₃H₂₅NO₂N,N-diethyl-2-hydroxy-6-(2- 347.189 348.245 >95 ¹H(naphthalen-2-yl)ethyl)benzamide [M + H ¹³C Sm19 C₂₀H₁₆O₃(E/Z)-2-methoxy-6-(2-(naphthalen-2- 304.110 305.219 92 E/Z ¹Hyl)vinyl)benzoic acid [M + H] ¹³C Sm20 C₁₉H₁₄O₃(E/Z)-2-hydroxy-6-(2(naphthalen-2- 290.094 289.079 91 E/Z ¹Hyl)vinyl)benzoic acid [M − H] ¹³C Sm21 C₁₅H₁₂O₃(E)-2-hydroxy-6-styrylbenzoic acid 240.079 239.067 >95 ¹H [M − H] ¹³CSm22 C₁₅H₁₄O₃ 2-hydroxy-6-phenethylbenzoic acid 242.094 241.100 >95 ¹H[M + H] Sm23 C₂₅H₂₇NO₃ (Z)-N,N,diethyl-2,4-dimethoxy-6-(2- 389.199390.222 >95 ¹H (naphthalen-2-yl)vinyl)benzamide [M + H] ¹³C Sm24C₂₅H₂₇NO₃ (E)-N,N,diethyl-2,4-dimethoxy-6-(2- 389.199 390.222 >95 ¹H(naphthalen-2-yl)vinyl)benzamide [M + H] ¹³C Sm25 C₂₅H₂₉NO₃N,N,diethyl-2,4-dimethoxy-6-(2- 391.215 392.240 >95 ¹H(naphthalen-2-yl)vinyl)benzamide ¹³C Sm26 C₂₃H₂₅NO₃N,N,diethyl-2,4-hydroxy-6-(2- 363.183 364.226 >95 ¹H(naphthalen-2-yl)ethyl)benzamide [M + H] ¹³C Sm27 C₂₁H₁₈O₄(E)-2,4-dimethoxy-6-(2-(naphthalen-2- 334.121 335.193 >95 ¹Hyl)vinyl)benzoic acid [M + H] Sm28 C₂₀H₁₆O₄(E/Z)-2-hydroxy-4-methoxy-6-(2- 320.105 319.093 91 E/Z ¹H(naphthalen-2-yl)vinyl)benzoic acid [M − H] ¹³C Sm29 C₂₀H₁₈O₄2-hydroxy-4-methoxy-6-(2(naphthalen- 322.121 321.120 90 ¹H2-yl)ethyl)benzoic acid [M − H] ¹³C Sm30 C₂₁H₂₀O₄2,4-dimethoxy-6-(2-(naphthalen-2- 336.136 337.143 >95 ¹Hyl)ethyl)benzoic acid Sm31 C₂₀H₁₅ClO₃ (E)-4-chloro-2-methoxy-6-(2-338.071 339.139 >95 ¹H (naphthalen-2-yl)vinyl)benzoic acid [M + H] ¹³CSm32 C₁₉H₁₃ClO₃ (E)-4-chloro-2-hydroxy-6-(2- 324.055 325.045 90 ¹H(naphthalen-2-yl)vinyl)benzoic acid [M + H] ¹³C Sm33 C₁₉H₁₅ClO₃(E)-4-chloro-2-hydroxy-6-(2- 326.071 325.062 85 ¹H(naphthalen-2-yl)ethyl)benzoic acid [M − H] Sm34 C₂₁H₁₈O₃(E)-2-methoxy-4-methyl-6-(2- 318.126 319.218 >95 ¹H(naphthalen-2-yl)vinyl)benzoic acid [M + H] ¹³C Sm35 C₂₀H₁₆O₃(E)-2-hydroxy-4-methyl-6-(2- 304.110 305.223 >95 ¹H(naphthalen-2-yl)vinyl)benzoic acid [M + H] ³C Sm36 C₂₀H₁₈O₃2-hydroxy-4-methyl-6-(2-(naphthalen- 306.126 307.235 90 ¹H2-yl)ethyl)benzoic acid [M − H] ¹³C Sm37 C₁₇H₁₈O₃(Z)-1,2,3-trimethoxy-5-styrylbenzene 270.126 271.136 >95 ¹H M + H ¹³CSm38 C₁₇H₁₈O₃ (E)-1,2,3-trimethoxy-5-styrylbenzene 270.136 271.136 >95¹H M + H ³C Sm39 C₁₇H₂₀O₃ 1,2,3-trimethoxy-5-phenethylbenzene 272.141273.150 >95 ¹H [M + H] ¹³C Sm40 C₁₄H₁₄O₃ 5-phenethylbenzene-1,2,3-triol230.094 n.d. >95 ¹H ¹³C Sm41 C₂₁H₂₀O₃ (Z)-2-(3,4,5- 320.141 321.149 >95¹H trimethoxystyryl)naphthalene [M + H] Sm42 C₂₁H₂₂O₃(E)-2-(2-(3,4,5-trimethoxycyclohexa- 322.157 321.149 >95 ¹H1,3-dien-1-yl)vinyl)naphthalene M + H ¹³C Sm43 C₂₁H₂₂O₃ 2-(3,4,5-322.157 323.164 >95 ¹H trimethoxyphenethyl)naphthalene [M + H] ¹³C Sm44C₁₈H₁₆0₃ 5-(2-(naphthalen-2-yl)ethyl)benzene- 280.110 n.d. >95 ¹H1,2,3-triol ¹³C Chemical analysis of compounds Sm15-Sm 44. Compoundpurity was determined by HPLC (ESI-MS/UV/ELSD) and their molecularformulae determined by HighRes MS (ESI microTOF-LC). ¹H and ¹³C NMRexperiments were used to confirm the structure (see Appendix A).

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Example 2 Pharmacological Targeting of the Transcription Factor SOX18Delays Breast Cancer in Mice Materials and Methods ExperimentalReproducibility

All data and statistical analysis in this study were generated from atleast three independent experiments unless indicated otherwise.Technical replicates were included in every experiment to reducebackground noise and detect technical anomalies. Samples of distinctexperimental conditions were not exposed to any specific method ofrandomization, and groups were assessed under non-blinded conditions.

Plasmid Preparation for Cell-Free Expression

The genetically encoded tags used here are enhanced GFP (GFP), mCherry(Cherry) and cMyc (myc). The proteins were cloned into the followingcell free expression Gateway destination vectors respectively:N-terminal GFP tagged (pCellFree_G03), N-terminal Cherry-cMyc(pCellFree_G07) and C-terminal Cherry-cMyc tagged (pCellFree_G08)(Gagoski et al. 2015). The Open Reading Frames (ORFs) corresponding tothe human SOX7 (BC071947), SOX17, RBPJ (BC020780) and MEF2C (BC026341)were sourced from the Human ORFeome collection version 1.1 and 5.1 orthe Human Orfeome collaboration OCAA collection (Open Biosystems) aspreviously described and cloned at the ARVEC facility, UQ DiamantinaInstitute. The entry clones pDONOR223 or pENTR201 vectors were exchangedwith the ccdB gene in the expression plasmid by LR recombination (LifeTechnologies, Australia). The full-length human SOX18 gene wassynthesized (IDT) and the transfers to vectors was realized usingGateway PCR cloning.

Cell-Free Protein Expression

The translation competent Leishmania tarentolae extract (LTE) wasprepared as previously described (Mureev et al. 2009, Kovtun et al.2011). Protein pairs were co-expressed by adding 30 nM of GFP templateplasmid and nM of Cherry template plasmid to LTE and incubating for 3hours at 27° C.

ALPHA-Screen Assay

The ALPHA-Screen Assay was performed as previously described (Siereckiet al. 2014), using the cMyc detection kit and Proxiplate-384 Plusplates (PerkinElmer). A serial dilution of each sample was measured. TheLTE lysate co-expressing the proteins of interest was diluted in bufferA (25 mM HEPES, 50 mM NaCl). For the assay, 12.5 μL (0.4 μg) ofAnti-cMyc coated Acceptor Beads in buffer B (25 mM HEPES, 50 mM NaCl,0.001% NP40, casein) were aliquoted into each well. This was followed bythe addition of 2 μL of diluted sample and 2 μL of biotin labeledGFP-Nanotrap in buffer A. The plate was incubated for 45 min at RT.Afterward, 2 μL (0.4 μg) of Streptavidin coated Donor Beads diluted inbuffer A, were added, followed by incubation in the dark for 45 min atRT. The ALPHA-Screen signal was obtained on an Envision Multilabel PlateReader (PerkinElmer), using the manufacturer's recommended settings(excitation: 680/30 nm for 0.18 s, emission: 570/100 nm after 37 ms).The resulting bell-shaped curve is an indication of a positiveinteraction, while a flat line reflects a lack of interaction betweenthe proteins. The measurement of each protein pair was repeated aminimum of three times using separate plates. The Binding Index wascalculated as: BI=[(I−I_neg)/(I_ref−I_neg)]×100

For each experiment, I is the highest signal level (top of the hookeffect curve) and Ineg is the lowest (background) signal level. Thesignals were normalized to the Iref signal obtained for the interactionof SOX18 with itself.

For PPI disruption assay, protein pairs expressed in LTE were incubatedfor 1 h with 100 μM Sm4 or DMSO alone (0.7% DMSO final). 100 μM Sm4 orDMSO was also added to buffer B. PPI disruption was calculated as:(1−I_Sm4/I_DMSO)×100.

For IC50 determination, the assay was identical but a dilution range ofSm4 was used (0.3 to 300 μM). Percentage of interaction was calculatedas: I_Sm4/1 DMSO×100. Data from at least 3 independent experiments werefitted in GraphPad Prism (RRID: SCR_007370) version 6.0 using3-parameter non-linear regression.

Cell Culture and Transfection

COS-7 cells were purchased from ATCC (CRL-1651, RRID: CVCL_0224)cultured at 37° C., 5% CO2 in DMEM (Life technologies, 11995) with addedFBS, sodium pyruvate, L-glutamine, penicillin, streptomycin,non-essential amino acids and HEPES(N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid). COS-7 cells weretransfected for 4-6 h, and incubated for another 24 h before lysis andluciferase assay (Perkin Elmer, 6016711). Human umbilical veinendothelial cells (HUVECs) were purchased from Lonza Australia(CC-2519A). HUVEC for ChIP-MS, ChIP-seq and RNA-seq analyses weretransfection for 7 h and incubated another 14 h. During small moleculetreatment, cells were grown in medium containing low serum (0.4% FBS).HUVECs were cultured at 37° C., 5% CO2 in EGM-2 media supplementedaccording to the EGM-2 bullet kit instruction (Lonza, CC-3162). Cellsfor were grown in 35 mm dishes to 80-90% confluency, and transfectedwith plasmid mouse pSG5 Sox18, plasmid pSG5 cMyc-Sox18, or plasmid cMycusing X-tremegene 9 DNA transfection reagent (Roche, 06365787001)according to the manufacturer's instructions. All cell lines were testednegative for mycoplasma contamination.

Chromatin Immunoprecipitation

ChIP experiments were performed as previously described (Schmidt et al.2009). Immunoprecipitation was performed using Anti-cMyc (CellSignaling, #2276, RRID: AB_2314825) on HUVECs overexpressing cMyc-taggedSOX18.

ChIP-Seq and Analysis

Following IP, DNA amplification was performed using TruSeq ChIPseq kit(Illumina, IP-202-1012), using 0.5 μM of the universal reverse PCRprimer and the forward PCR primer containing the index sequence ofchoice in 50 μL 1×NEBNext High-Fidelity PCR Master Mix (New EnglandBiolabs, M0541). The number of PCR cycles ranged from 13 to 18,depending on the ChIP efficiency. The PCR product was purified usingAMPure beads (1.8 volume) and eluted in 20 μL of resuspension buffer(Tris-Acetate 10 mM pH 8). The library was quantified using the KAPAlibrary quantification kit for Illumina sequencing platforms (KAPABiosystems, KK4824) and 50 bp single end reads were sequenced on aHiSeq2500 following the manufacturer's protocol. Illumina fastq fileswere mapped to the GRCh37/UCSC hg19 genome assembly using bowtie, andpeaks were called using MACS version 2.1.0. using input. To avoid falsepositive peaks calling due to the cMyc epitope, ChIP-seq with the cMycepitope only were performed in parallel to SOX18-cMyc ChIP-seq and peakscalled in these experimental conditions were substracted to the peakscalled in the SOX18-cMyc conditions. Genomic Regions Enrichment ofAnnotations Tool (GREAT, RRID: SCR_005807)) was used to analyse thefunctional significance of cis-regulatory regions. ChIP-seq data areavailable in the ArrayExpress database (www.ebi.ac.uk/arrayexpress,RRID: SCR_002964) under accession number E-MTAB-4480 (SOX7) andE-MTAB-4481 (SOX18).

Chip-Ms (Rime)

ChIP-MS experiments were performed as previously described (Mohammed etal. 2013). Peptides common between SOX18-cMyc and the negative control(cMyc-only) were binned and only peptides that were uniquely detected inthe SOX18-cMyc transfected cell were considered for analysis.

RNA-Seq and Analysis

Quadruplicate samples were processed for whole transcriptome sequencingusing TruSeq stranded total RNA library prep kit (Illumina). Reads weremapped to the hg19 reference human genome using STAR aligner (Dobin etal. 2013), and only uniquely aligned reads were considered. Transcriptswere assigned to genes using htseq_count (HTseq package) (Anders, Pyl,and Huber 2015), and differential expression was calculated using DEseq2(Love, Huber, and Anders 2014). Genes with adjusted p-value <0.05 wereconsidered significant.

Differentially expressed genes were identified between Sm4-treated andDMSO control in SOX18 over-expressing cells, and separated inup-regulated and down-regulated (DOWN) genes. The locations of theirtranscription start sites (TSS) were correlated to the locations oftranscription factors binding events that are available from the ENCODEconsortium (RRID: SCR_006793), and from the SOX18 and SOX7 ChIP-seqexperiment we performed in this study. To ensure that the TSSs wereindependent, a TSS was allowed to only be assigned to 1 ChIP-seq peak.Transcripts with 2-fold absolute fold change (log 2FC≥1 or ≤−1) wereincluded for distance to TSS analysis. The median distance between theTSSs and binding events was compared to the expected distance of a setof randomly selected genes to obtain the median ratio. The control setof genes was selected from the pool of genes expressed in HUVECs so thatthey had a similar distribution of expression levels. To ensure that nobias was introduced by potential co-regulation of genes by SOX18 and anyother transcription factor analysed, we subtracted genes with SOX18peaks from the analyses for other transcription factors. The reverseanalysis was also performed, subtracting genes containing c-JUN peaksfrom the analysis for SOX18. RNA-seq data are available in theArrayExpress database (www.ebi.ac.uk/arrayexpress) under accessionnumber E-MTAB-4511.

Quantitative RT-PCR

Total RNA was extracted using RNeasy mini kit (Qiagen, 74106) accordingto the manufacturers protocol, including on column DNA digestion. cDNAwas synthetized from 1 μg of purified RNA using the high capacity cDNAreverse transcription kit (Life Technologies, 4368813). Amplificationand quantitation of target cDNA was performed in technical triplicate ofat least 3 biological replicates using the SYBR green (LifeTechnologies, 4312704) methods. Reactions were run in 10 μL in 384-wellplates using the ViiA 7 Real-Time PCR system. Housekeeper genes (β-actinfor tg(Dll4 in3:eGFP), ef1α for tg(−6.5kdrl:eGFP), chd5 fortg(flila:eGFP, −6.5kdrl:mCherry), RPL13 and GAPDH for HUVECs) wereselected based on the stability of their expression throughout the setof experimental conditions, or chosen on grounds of their vascularexpression to normalize to endothelial cell content. Primer efficiencieswere calculated using LinRegPCR, and amplification data was analysedusing ViiA7 software and the Q-gene PCR analysis template.

Zebrafish Aquaculture and Analysis

Zebrafish were maintained as previously described (Hogan et al. 2009),and all procedures involving animals conformed to guidelines of theanimal ethics committee at the University of Queensland(IMB/030/16/NHMRC/ARC/HF) or were approved by local ethical review andlicensed by the UK Home Office (PPL 30/2783 and PPL 30/3324). Thetg(−6.5kdrl:eGFP), tg(fli1a:eGFP,−6.5kdrl:mCherry) and tg(Dll4 in3:GFP)were previously described (Sacilotto et al. 2013, Duong et al. 2014,Lawson and Weinstein 2002).

Dechlorination was performed by treatment with 25 μg/mL or 5 μg/mLpronase for 2 h, or overnight, respectively. Zebrafish larvae wereanesthetized using 0.01% tricaine. Representative larvae were embeddedin low-melting point agarose and imaged with the Zeiss LSM 710 confocalmicroscope.

Zebrafish in Situ Hybridization and Sectional Analysis

Wholemount zebrafish (28 and 48 hpf) in situ hybridization was performedas previously described (Thisse and Thisse 2008) with probe templatesfor dab (Song et al. 2004) and ephrinB2a (Durbin et al. 1998). Yolk sacwas removed prior to addition of in 70% glycerol. For transversesections, whole larvae where embedded in 4% agarose, sectioned at 150 μmusing the Leica VT1000 S vibrating microtome. Imaging was performed onthe Olympus BX-51 brightfield microscope (ISH), and Zeiss LSM 510confocal microscope. For fluorescent images, larvae were DAPI-stainedbefore embedding.

Small Molecule Treatment and Morpholino Injections

All treatment with putative small molecule inhibitors, and correspondingcontrol conditions, were performed in the presence of low concentrationof DMSO (1% v/v) to achieve reliable homogeneous solutions, and wereprepared from 10 mM DMSO stock. For cell culture, small molecules wereadded to fresh media directly following transfection and cells weregrown in this media until time-point of cell harvesting. For in vivoexperiments involving zebrafish, compound treatment was initiated at thedesignated timepoints by replacing the media, and media+compound wasrefreshed daily for the duration of the experiment. PTU treatment(0.003%) was done in parallel with the small molecules to block pigmentformation when necessary. Previously published and validated morpholinooligomers against sox7, sox18 (Herpers et al. 2008) and rbpj (Sacilottoet al. 2013) were micro-injected into single cell zebrafish zygotes at 5ng for experiments performed with tg(6.5kdrl:eGFP) andtg(fli1a:eGFP,−6.5kdrl:mCherry), and 0.125-0.15 pmol suboptimalconcentrations for experiments performed with tg(Dll4 in3:eGFP).

Mice and Mouse Model

BALB/c wild-type (WT) were purchased from Walter and Eliza HallInstitute for Medical Research and used between the ages of 6 and 10weeks. Mouse 4T1.2 mammary carcinoma cells were cultured in completeRPMI with 10% FBS in a 5% CO2 incubator. 5×104 4T1.2 tumor cells wereinoculated into the fourth mammary fat-pad of BALB/c WT mice aspreviously described (Mittal et al. 2014). Briefly, on day 3 after tumorimplantation, mice were orally gavaged daily for 10 days with 25 mg/kgof body weight Sm4, aspirin or vehicle PBS. Tumor size was measured witha digital caliper as the product of two perpendicular diameters. Bloodplasma was collected from mice on day 7 and 12, and Sm4 concentrationswere analyzed using a 4000 Qtrap LC-MS/MS system mass spectrometer. Onday 12, mice were anesthetized to surgically remove primary tumor, ormice were put through surgery procedure with no excision of the primarytumor, and the wound was closed with surgical clips. Tumors werecollected in formalin for histology. Lungs were harvested on day 28 andfixed in Bouin's solution for 24 h and metastatic tumor nodules werecounted under a dissection microscope. Survival of the mice wasmonitored in experiments where the lungs were not harvested. Groups of 6to 14 mice per experiment were used for experimental tumor assays, toensure adequate power to detect biological differences. All experimentswere approved by the QIMR Berghofer Medical Research Institute AnimalEthics Committee (P1505).

For quantitation of the vasculature in the tumors, fixed tissues wereembedded in 4% agarose and sectioned all the way through at 300 μm on aLeica VT1000 S vibrating microtome. Sections were collected on glassslides and imaged for bright field analysis on the penetration ofperfused vessels. Subsequently, immunofluorescent staining was performedon sections using anti-mouse Endomucin (cat #sc-53941, RRID:AB_2100038), ERG (cat #ab92513, RRID: AB_2630401), PROX1 (AngioBio cat#11-002, RRID: AB_10013720) and Podoplanin (AngioBio cat #11-033,AB_2631191) antibodies. Whole tumor sections were imaged by acquiring aseries of images along the z-axis using a 10× objective on a Zeiss LSM710 confocal microscope. Subsequently, high-resolution images werecaptured using a 20× objective on 3-4 separate regions from each tumor,to account for heterogeneity of the vascular density within the tumorsand minimise bias. Raw image files with identical dimensions (1274.87μm×1274.87 μm×89.05 μm) were loaded into Imaris (Bitplane, RRID:SCR_007370), and processed using “spots” function to count ERG orPROX1-positive nuclei and “surface” to calculate volume or area ofEndomucin or Podoplanin positive vessels. For each tumor (n=6), countsfrom the multiple regions were averaged and the data was plotted inGraphpad Prism 6.

BALB/c wild-type (WT) were purchased from Walter and Eliza HallInstitute for Medical Research and used between the ages of 6 and 10weeks. Mouse 4T1.2 mammary carcinoma cells were cultured in completeRPMI with 10% FBS in a 5% CO2 incubator. 5×104 4T1.2 tumour cells wereinoculated into the fourth mammary fat-pad of BALB/c WT mice aspreviously described (Mittal et al. 2014). Briefly, on day 3 aftertumour implantation, mice were orally gavaged daily for 10 days withdifferent doses of Sm4 ranging from 5 mg/kg to of body weight or vehiclePBS. Tumour size was measured with a digital caliper as the product oftwo perpendicular diameters. On day 12, mice were anesthetized tosurgically remove primary tumour, and the wound was closed with surgicalclips. Tumours were collected in formalin for histology. Survival of themice was monitored in groups of 6 to 12 mice per experiment, to ensureadequate power to detect biological differences. All experiments wereapproved by the QIMR Berghofer Medical Research Institute Animal EthicsCommittee (P1505).

Results and Discussion

SOX proteins activate individual target genes by recruiting specificinteracting partners (Sarkar and Hochedlinger 2013), but only twoprotein-protein interactions for the SOXF group (SOX18-MEF2C andSOX17-OCT4) have been identified to date (Hosking et al. 2001, Jauch etal. 2011). We first mapped the SOX18 interactome (the network of SOX18interacting partners), using a combination of unbiased proteomictechnologies. Chromatin immunoprecipitation coupled to mass spectrometry(ChIP-MS) provided a first-pass screen for proteins associated withchromatin-bound SOX18 in human umbilical vein endothelial cells (HUVECs)(Mohammed et al. 2013), then, ALPHA-Screen resolved SOX18-dependentcomplexes into pairwise interactions using in vitro translatedfull-length proteins (FIG. 9A) (Mureev et al. 2009, Kovtun et al. 2011,Sierecki et al. 2013, Sierecki et al. 2014, Gambin et al. 2014). ChIP-MSanalysis revealed 289 proteins, representing a variety of gene ontology(GO) classes of molecular function, that associate directly orindirectly with SOX18 (FIGS. 9B and 10A-C). To increase our chance ofidentifying direct interactors, we focused on proteins known to benucleic acid and/or protein binding (FIG. 9B, purple). From this subset,we chose 8 known transcription factors, helicases, co-repressors, RNAbinding and DNA-repair molecules (FIG. 10A,B). Using ALPHA-Screen, weobserved that SOX18 interacts with itself, and also forms pairwiseinteractions with DDX1, DDX17, ILF3, STAT1, TRIM28, and XRCC5 (FIG. 9C,left column ‘+’, and FIG. 10D).

In addition, we studied potential pairwise interactions of 6 well-knownTFs able to regulate endothelial cell function (ESR1, NR2F2, RBPJ, SOX7,SOX17 and CTNNB1), and the only identified SOX18 protein partner MEF2C(Hosking et al. 2001). The well-characterized SOX9 homo-dimer (Bernardet al. 2003) was included as a positive control to validate theALPHA-Screen signal (FIG. 10D). SOX18 was found to interact with allendothelial transcription factors tested, with the possible exception ofSOX17 and CTNNB1, which showed a binding affinity below the arbitrarythreshold (FIG. 9C, ‘−’).

Having identified an array of proteins able to interact with SOX18, wethen went on to test the activity of a small-molecule compound, Sm4(Figure on these interactions. Sm4, derived from a natural product foundin the brown alga Caulocystis cephalornithos, was identified in ahigh-throughput screen for potential SOX18 blockers (see Example 1). Wefound that Sm4 significantly disrupted 6 out of the 12 validated SOX18interactions (FIG. 9C, right column), with IC50 values ranging from 3.3μM for SOX18-SOX18 to 65.9 μM for SOX18-RBPJ dimers (FIGS. 9D and 10F).To assess a differential effect of Sm4 on the distinct SOXF members, weexplored an additional set of PPIs between all three SOXF proteins andMEF2C, RBPJ and OCT4 (FIG. 11 ). Like SOX18, SOX7 is able to interactwith RBPJ and SOX18 itself, both of which interactions are at leastpartially disrupted by Sm4. We further found that all three SOXFproteins can form a heterodimer with OCT4, whereas only the SOX17-OCT4interaction is affected by Sm4. Importantly, neither SOX7 nor SOX17 havethe capacity to form a homodimer, and thus this component of Sm4 mode ofaction is highly specific to SOX18-SOX18 interaction. Furthercorroborating this, SOX9 homodimerization was unperturbed by Sm4 at upto 200 μM (FIGS. 9C-D and 10D). These results show that Sm4 selectivityleans towards a subset of SOX18-associated PPIs, but has the capabilityto interfere with SOX7 or SOX17 protein partner recruitment. Thisfeature of Sm4 is potentially advantageous in preventing SOXF redundancymechanism (Hosking et al. 2009, Kim et al. 2016).

To assess how SOX18 PPI disruption translates into transcriptionaldysregulation, we next performed a combination of genome-wide RNA-seqand ChIP-seq analyses in HUVECs. The most common binding motifidentified from the SOX18 ChIP-seq peaks corresponds to the previouslyreported SOX motif 5′-AACAAT-3′ (FIG. 13A) and the validity of thisChIP-seq dataset was further confirmed by GO term analysis andidentification of known SOX18 target genes such as Prox1 and Vcam1 (FIG.13B) (Francois et al. 2008, Hosking et al. 2004). We compared the globaltranscriptional effect of Sm4 treatment to DMSO control in SOX18overexpressing cells (FIG. 13C-E), and overlaid this list ofdifferentially expressed genes with the SOX18 ChIP-seq dataset. Usingthis overlay, we calculated the distance between the transcription startsite (TSS) of a gene and a TF binding event, as a proxy for thelikelihood of direct transcriptional regulation. To be able to analysehow this distance is altered by Sm4, we established a reference distancebetween the TSS of a random gene set and SOX18 binding events (FIG.12A). In parallel, we performed the same analysis for SOX7 (generatedin-house), and for all 7 transcriptional regulators available from theENCODE consortium (GATA2, c-FOS, c-JUN, CTCF, EZH2, MAX, c-MYC). Thisallowed us to distinguish between transcriptional targeting of SOX18 andpotential off target effects on other endothelial specific transcriptionfactors.

The cumulative SOX18 peak-to-TSS distance demonstrated that, overall,SOX18 peaks are 3.6 fold closer (p-value <0.001) to the TSS of Sm4down-regulated genes than to randomly distributed TSSs (FIG. 12B, topleft). These results are an indirect indication that the Sm4 affectedgenes are dysregulated through a specific effect on SOX18transcriptional activity. This correlation was not observed for 7 of theother transcription factors tested (FIGS. 12B and 13F), signifying thatSm4 does not have an off-target effect on these TFs activity.Interestingly, the TSS of Sm4 down-regulated genes were 2.05 fold closerto c-JUN binding events (p-value=0.011, Supplementary file 1c). Althoughonly mildly significant, this could suggest possible co-regulation bySOX18 and c-JUN on this subset of Sm4 down-regulated genes. Indeed,analysis of known motifs in SOX18 ChIP-seq peaks revealed anover-representation of c-JUN binding motifs (3.23% of SOX18 peaks,p-value=le-302) and ALPHA-Screen analysis further established that SOX18and c-JUN could physically interact (FIG. 14 ). We found that theexpression levels of the other TFs tested were unaltered by Sm4treatment. This is an important observation because it demonstrates thatthere was no bias introduced by an off-target modulation of thetranscript levels for these transcription factors in presence of Sm4.

To address the issue of potential transcriptional off-target effects ofSm4 on SOX TF family members we focused on closely related SOXF and SOXEproteins. Sm4 did not affect the transcriptional activity of eitherSOX17 or SOX9 proteins at any tested concentration (≤50 μM) incell-based reporter assays (FIG. 15 ) (Robinson et al. 2014, Lefebvre etal. 1997). Together, these results provide strong evidence that Sm4selectively targets SOX18-mediated transcription over other keyendothelial transcription factors and SOX proteins.

To investigate whether Sm4 is also able to perturb Sox18 transcriptionalactivation in vivo, we used the tg(−6.5kdrl:eGFP) transgenic zebrafishreporter line, previously validated as a readout for the combinedactivity of Sox7 and Sox18 (Duong et al. 2014). We treated these larvaeat 20 hours post fertilization (hpf) and observed that Sm4 treatmentsignificantly reduced SOX18-dependent egfp transcript levels (61%),similar to the effects of combined sox7/18 depletion using morpholinooligonucleotides (MO) (FIG. 16A,B). Importantly, these zebrafish embryosdeveloped normally and we found no evidence of toxicity.

We then used a second transgenic zebrafish reporter line tg(Dll4in3:eGFP), which harbours a regulatory element located in the intron 3of d114 gene. The activity of this Dll4 in3 enhancer does not fullyrecapitulate the endogenous d114 expression (Wythe et al. 2013,Sacilotto et al. 2013) (Wythe et al 2013 and Sacilotto et al 2013), butit does provide a useful tool to study the combinatorial activity ofSox7, Sox18 and the Notch effector Rbpj. Combined genetic interferencewith sox7, sox18 and rbpj has been shown to abolish Dll4 in3 activation,while single or double MO knockdowns have a much milder effect(Sacilotto et al. 2013). This mild repressive effect was recapitulatedby treatment with Sm4 alone (FIG. 16C,D). In addition, when rbpj MOinjections at suboptimal dose were combined with Sm4 treatment, therepressive effect was significantly increased by 11.5% (FIG. 16C,D).These data show that Sm4 interferes with Sox7/18 and Rbpj co-ordinatedactivation of the Dll4 in3 enhancer. As a negative control in vivo, weused the Sox9-dependent tg(col2a1:yfp) reporter line, and observed thatcontinuous Sm4 treatment between 2 and 6 days post fertilization did notperturb the transcriptional activity of Sox9 or the process ofchondrogenesis (FIG. 17 ). Together, this supports the proposedmechanism of action for Sm4 as a selective SOX18 inhibitor in vivo.

To further demonstrate the small molecule inhibition of Sox18 functionin vivo, we next investigated whether Sm4 treatment would be able tocause a vascular phenotype, similar to that of sox7/sox18 geneticallydisrupted zebrafish (Hermkens et al. 2015). This phenotype ischaracterised by an arteriovenous specification defect, with reducedexpression levels of arterial markers (Cermenati et al. 2008, Herpers etal. 2008, Pendeville et al. 2008). We treated zebrafish larvaeharbouring the arterial/venous reporter tg(fli1a:eGFP,−6.5kdrl:mCherry)with 1.5 μM Sm4 during the relevant developmental window, starting from16 hpf (FIG. 18A). These larvae acquired an enlarged posterior cardinalvein (PCV) at the expense of the dorsal aorta (DA) (FIGS. 16E-G and18B), with arteriovenous shunts and incomplete trunk circulation (FIG.18C,D). qRT-PCR analysis of blood vascular markers at 24 and 48 hpfrevealed a significant dysregulation of arterial and venous genes inSm4-treated conditions compared to DMSO, particularly efnb2a, hey1 andefnb4a (FIGS. 16H and 18E).

Due to SoxF redundancy in arteriovenous specification, an A/Vmalformation phenotype is typically only observed in double loss of Sox7and Sox18 function. Since Sm4 appeared to partially interfere withSox7-Rbpj and Sox7-Sox18 PPIs in vitro, we turned to a Sox7 specificphenotype to assess whether this TF activity was inhibited by Sm4 invivo. The hallmark of sox7 genetic disruption is a short circulatoryloop in the head formed by the lateral dorsal artery (Mohammed et al.2013), resulting in perturbed facial circulation (Hermkens et al. 2015).In presence of Sm4, we observe minor malformation to the LDA reminiscentof a partial Sox7 loss of function phenotype. However, the bloodcirculation in the head is unaffected in Sm4-treated larvae, signifyingthat a short circulatory loop has not fully formed. This phenotypesupports of the conclusion that Sox7 activity is only partially affectedin presence of the small compound. Overall, these results are congruentwith the genome-wide inhibitory effects observed in vitro, demonstratingthat Sm4 selectively interfered with the transcriptional activity ofSox18 and SoxF-mediated vascular formation in vivo.

As a final demonstration of the anti-angiogenic potential of Sm4 in atherapeutically relevant setting, we next assessed its efficacy in apreclinical model of breast cancer. BALB/c mice were inoculated withhighly metastatic 4T1.2 mammary carcinoma cells into the mammary fatpad, and 3 days were allowed for the engraftment of the tumor, afterwhich treatment was initiated with either 25 mg/kg/day of Sm4, aspirinor vehicle PBS (FIG. 19A). Aspirin was chosen as a negative controlbecause of the structural similarity to Sm4. Daily treatment wasmaintained for a duration of 10 days, after which the primary tumor wasresected and effects on disease latency were monitored (FIG. 19A). As anindirect indication of target engagement, we first confirmed theexpression of Sox18 in the 4T1.2 tumor vasculature by in situhybridization (FIG. 19B). We next went on to measure Sm4 bioavailabilityduring the course of the treatment. Sm4 was consistently detected inblood plasma at 2 different time points, with a mean concentrationincreasing over time from 38.3 μg/mL to 55.2 μg/mL (FIG. 19C).

PBS vehicle- or aspirin-treated mice succumbed to the 4T1.2 tumor burdenwith a median latency of 33 and 34 days respectively (FIG. 19D), whereasSm4-treated mice had a significant increase in their overall survivalwith a median latency of 44 days (p-value <0.01). As shown in theassessment of Sm4 dose response of FIG. 201 , increasing theconcentration of Sm4 resulted in further improvements to overallsurvival of 4T1.2 inoculated mice. By way of example, treatment of micewith 50 mg/kg Sm4 resulted in a median latency of 73 days versus amedian latency of 40 days for vehicle treated mice.

To further investigate what could cause such an effect, the size of thetumors was monitored during the treatment, as well as the formation ofspontaneous lung metastases. While the size of the primary tumor wasunchanged by Sm4 treatment (FIG. 19E), we found a 67% reduction in themean number of lung metastases at day 28 after tumor inoculation (FIG.19F).

In order to establish a correlation between the metastatic rate and atumor induced vascular response, we investigated the blood vesseldensity in the intra-tumoral and peri-tumoral regions (FIGS. 19G and 20). Whole tumors were sectioned, and brightfield microscopy revealed anoverall reduction in blood vessel coverage, as indicated by the presenceof red blood cells (FIG. 19G, asterisks). Further analysis usingimmunofluorescent staining for endothelial cell markers ERG (nuclear)and Endomucin (EMCN, membranous), showed a significant decrease in thenumber of endothelial cells (48%, p-value <0.05), as well as the volumeof the blood vessels (55%, p-value <0.01) in the tumors of Sm4-treatedmice (FIGS. 19H,I and 21). Using lymphatic specific markers PROX1 andpodoplanin (PDPN), we also assessed the effect of Sm4 on the tumorinduced lymphangiogenic response, and found that the density of thetumor associated lymphatic vessels was greatly reduced (65%, p-value<0.01) in treated conditions, as well as the number of lymphaticendothelial cells (70%, p-value <0.001) (FIG. 22 ). This lymphaticresponse to Sm4-treatment is consistent with that of SOX18 loss offunction during lymphatic spread of solid cancers (Duong et al. 2012)Together, this demonstrates that Sm4 improved the outcome of inducedbreast cancer by interfering with tumor-induced neovascularization andassociated metastasis.

Induction of angio- and lymphangiogenesis is a hallmark of solid cancer,and is a critical step towards enabling tumor metastatic dissemination.Conventional approaches to target transcription factors have focused oninterfering with oncogenes that are dysregulated to promote tumor celltransformation (Gormally et al. 2014, Illendula et al. 2015, Moelleringet al. 2009, Zhang et al. 2012). Here, we validate a novel complementarystrategy that relies on targeting a developmental transcription factorfrom the host vasculature that can facilitate metastatic spread. Ourresults provide a proof of concept that targeting the transcriptionfactor SOX18 with Sm4 is an effective molecular strategy to interferewith the metastatic spread in a pre-clinical model of breast cancer.

Throughout the specification the aim has been to describe the preferredembodiments of the invention without limiting the invention to any oneembodiment or specific collection of features. It will therefore beappreciated by those of skill in the art that, in light of the instantdisclosure, various modifications and changes can be made in theparticular embodiments exemplified without departing from the scope ofthe present invention.

All computer programs, algorithms, patent and scientific literaturereferred to herein is incorporated herein by reference.

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1. A method of treating cancer comprising administering to a subject aneffective amount of a compound of Formula 1:

wherein, R₁ is selected from the group consisting of OH and OR₆ whereinR₆ is C₁₋₄alkyl; R₂ is selected from the group consisting of H, COORS,and C(O)NR₈R₉ wherein R₇, R₈ and R₉ are independently selected from thegroup consisting of H and C₁₋₄ alkyl; R₃ is L-A wherein L is a linkerselected from the group consisting of C₂₋₈alkyl, C₂₋₈ alkenyl and C₂₋₈alkoxyalkyl and A is an optionally substituted naphthyl; R₄ is selectedfrom the group consisting of H, OR₁₀, halo and C₁₋₄ alkyl wherein R₁₀ isselected from the group consisting of H and C₁₋₄ alkyl; and R₅ isselected from the group consisting of H, OR₁₁, halo and C₁₋₄ alkylwherein R₁₁ is selected from the group consisting of H and C₁₋₄ alkyl;or a pharmaceutically effective salt, solvate or prodrug thereof.
 2. Themethod according to claim 1, wherein the cancer is an angiogenesisand/or lymphangiogenesis related cancer.
 3. The method according toclaim 1 wherein the cancer is a SOX7, SOX17, or SOX18-dependent cancer.4. The method according to claim 1, wherein the cancer is selected fromthe group consisting of sarcomas, carcinomas, lymphomas, leukemias, andblastomas.
 5. The method according to claim 4, wherein the cancer isselected from the group consisting of prostate cancer, breast cancer,lung cancer, bladder cancer, renal cancer, ovarian cancer, cervicalcancer, uterine cancer, colon cancer, colorectal cancer, gastric cancer,head and neck cancer, liver cancer, kidney cancer, skin cancer,pancreatic cancer, pituitary cancer, musculoskeletal cancer, hemangioma,angioma, angiosarcoma, hepatoma, and hepatocellular carcinoma.
 6. Themethod according to claim 1 wherein cancer metastasis is inhibited orprevented.
 7. The method according to claim 1 wherein the compound ofFormula I selectively inhibits SOX18-mediated transcription.
 8. Themethod according to claim 1 wherein the compound of Formula I inhibitsboth DNA binding and transcription factor protein partner recruitment ofSOX18.
 9. The method according to claim 1, wherein the compound ofFormula I inhibits SOX18 homodimerization.
 10. The method according toclaim 1, wherein the compound of Formula I inhibits SOX18-RBPJheterodimerisation.
 11. The method according to claim 1, wherein for thecompound of Formula 1: R₁ is selected from the group consisting of OHand OR₆ wherein R₆ is C₁₋₄alkyl; R₂ is selected from the groupconsisting of H, COOR₇, and C(O)NR₈R₉ wherein R₇, R₈ and R₉ areindependently selected from H and C₁₋₄ alkyl; R₃ is L-A wherein L is alinker selected from the group consisting of C₂₋₈alkyl, C₂₋₈ alkenyl andC₂₋₈ alkoxyalkyl and A is an optionally substituted naphthyl, andwherein the substituted groups are selected from the group consisting ofhalo, C₁₋₄ alkyl, OC₁₋₄ alkyl, NH₂, NH(C₁₋₄ alkyl), and N(C₁₋₄ alkyl)₂;R₄ is selected from the group consisting of H, OR₁₀, halo and C₁₋₄ alkylwherein R₁₀ is selected from H and C₁₋₄ alkyl; and R₅ is selected fromthe group consisting of H, OR₁₁, halo and C₁₋₄ alkyl wherein R₁₁ isselected from the group consisting of H and C₁₋₄ alkyl.
 12. The methodaccording to claim 1, wherein for the compound of Formula 1: R₁ isselected from the group consisting of OH and OR₆ wherein R₆ isC₁₋₄alkyl; R₂ is selected from the group consisting of COOR₇ andC(O)NR₈R₉ wherein R₇, R₈ and R₉ are independently selected from thegroup consisting of H and C₁₋₄ alkyl; R₃ is L-A wherein L is a linkerselected from the group consisting of C₂₋₈alkyl, C₂₋₈ alkenyl and C₂₋₈alkoxyalkyl and A is an optionally substituted naphthyl; R₄ is selectedfrom the group consisting of H, OR₁₀, halo and C₁₋₄ alkyl wherein R₁₀ isselected from the group consisting of H and C₁₋₄ alkyl; and R₅ is H. 13.The method according to claim 1, wherein for the compound of Formula 1:R₁ is selected from the group consisting of OH and OR₆ wherein R₆ isC₁₋₄alkyl; R₂ is selected from the group consisting of H, COOR₇, andC(O)NR₈R₉ wherein R₇, R₈ and R₉ are independently selected from thegroup consisting of H and C₁₋₄ alkyl; R₃ is L-A wherein L is a linkerselected from the group consisting of C₂₋₈alkyl, C₂₋₈alkenyl and C₂₋₈alkoxyalkyl and A is selected from the group consisting of anunsubstituted naphthyl or a naphthyl substituted with one or more groupsselected from the group consisting of halo, C₁₋₄ alkyl, OC₁₋₄alkyl, NH₂,NH(C₁₋₄alkyl), and N(C₁₋₄ alkyl)₂; R₄ is selected from the groupconsisting of H, OR₁₀, halo and C₁₋₄ alkyl wherein R₁₀ is selected fromthe group consisting of H and C₁₋₄ alkyl; and R₅ is selected from thegroup consisting of H, OR₁₁, halo and C₁₋₄ alkyl wherein R₁₁ is selectedfrom the group consisting of H and C₁₋₄ alkyl.
 14. The method accordingto claim 1, wherein for the compound of Formula 1: R₁ is selected fromthe group consisting of OH and OR₆ wherein R₆ is C₁₋₄alkyl; R₂ isselected from the group consisting of COOR₇ and C(O)NR₈R₉ wherein R₇, R₈and R₉ are independently selected from the group consisting of H andC₁₋₄ alkyl; R₃ is L-A wherein L is a linker selected from the groupconsisting of C₂₋₈alkyl, C₂₋₈ alkenyl and C₂₋₈ alkoxyalkyl and A is anoptionally substituted naphthyl; R₄ is H; and R₅ is H.
 15. The methodaccording to claim 1, wherein for the compound of Formula 1: R₁ is OH;R₂ is COORS, wherein R₇ is selected from the group consisting of H andC₁₋₄alkyl; R₃ is L-A wherein L is a linker selected from the groupconsisting of C₂₋₈alkyl, C₂₋₈ alkenyl and C₂₋₈ alkoxyalkyl and A is anoptionally substituted naphthyl; R₄ is H; and R₅ is H.
 16. The methodaccording to claim 1, wherein for the compound of Formula 1: R₁ is OH;R₂ is COOH; R₃ is L-A wherein L is a linker selected from the groupconsisting of C₂₋₈alkyl, C₂₋₈ alkenyl and C₂₋₈ alkoxyalkyl and A is anoptionally substituted naphthyl; R₄ is H; and R₅ is H.
 17. The methodaccording to claim 1, wherein for the compound of Formula 1: R₁ is OH;R₂ is COOH; R₃ is L-A wherein L is C₂₋₈ alkyl and A is an optionallysubstituted naphthyl; R₄ is H; and R₅ is H.
 18. The method according toclaim 1, wherein for the compound of Formula 1: R₁ is OH; R₂ is COOH; R₃is L-A wherein L is C₂₋₈ alkyl and A is an unsubstituted naphthyl or anaphthyl substituted with one or more groups selected from the groupconsisting of halo, C₁₋₄ alkyl, OC₁₋₄ alkyl, NH₂, NH(C₁₋₄ alkyl), andN(C₁₋₄ alkyl)₂; R₄ is H; and R₅ IS H.